Antibodies to human complement factor c2b and methods of use

ABSTRACT

Provided are antibodies and antigen-binding fragments thereof that bind specifically to human complement factor C2 and are capable of inhibiting activation of the classical and lectin pathways of the complement system. The antibodies and antigen-binding fragment exhibit improved manufacturability, pharmacokinetics, and antigen sweeping. Also provided are pharmaceutical compositions comprising the antibodies and antigen-binding fragments, nucleic acids and vectors encoding the antibodies and antigen-binding fragments, host cells comprising the nucleic acids or vectors, and methods of making and using the antibodies and antigen-binding fragments. The antibodies and antigen-binding fragments can be used to inhibit the classical pathway of complement activation in a subject, e.g., a human. The antibodies and antigen-binding fragments can also be used to inhibit the lectin pathway of complement activation in a subject, e.g., a human.

RELATED APPLICATION

This application claims benefit of priority from U.S. Provisional PatentApplication No. 62/779,102, filed Dec. 13, 2018, the entire content ofwhich is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 13, 2019, isnamed 618637_AGX5-048_ST25.txt and is 94,280 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the fields of immunology and molecularbiology. More particularly, the present invention relates tocompositions and methods for inhibiting the activation of the classicaland lectin pathways of the complement system and use thereof in thetreatment of human conditions. The invention in particular relates tobinding molecules that bind to human complement factor C2 and methods ofmaking and using same.

BACKGROUND OF THE INVENTION

The complement system involves a cascading series of plasma enzymes,regulatory proteins, and proteins capable of cell lysis. Prior toactivation, various complement factors circulate as inactive precursorproteins. Activation of the system leads to an activation cascade whereone factor activates the subsequent one by specific proteolysis ofcomplement protein further downstream in the cascade.

Activation of the complement system can occur via three pathways, theclassical (or classic) pathway, the alternative pathway, and the lectinpathway. The classical pathway is activated by interaction of antigenand IgM, IgG1, IgG2, or IgG3 antibody to form immune complexes that bindC1q, a subunit of complement component C1. The alternative pathway isactivated by IgA-containing immune complexes or recognition of bacteriaand other activating surfaces. The lectin pathway is responsible for anantibody-independent pathway of complement activation that is initiatedby binding of mannan-binding lectin (MBL), also known as mannose-bindinglectin or mannan-binding protein (MBP), to certain carbohydrates on thesurface of a variety of pathogens.

Activation of the classical pathway begins with sequential activation ofC1, C4, and C2; C2 is in turn cleaved into C2a and C2b. Activation ofthe alternative pathway begins with sequential activation of complementcomponents D, C3, and B. Each pathway cleaves and activates a commoncentral component, C3 or the third complement factor, which results inthe activation of a common terminal pathway leading to the formation ofthe membrane-attack complex (MAC, comprising complement componentsC5b-9; Muller-Eberhard, Annu Rev Biochem 1988, 57:321). Duringcomplement activation, several inflammatory peptides like theanaphylatoxins C3a and C5a are generated as well as the MAC. Theseactivation products elicit pleiotropic biological effects such aschemotaxis of leukocytes, degranulation of phagocytic cells, mast cellsand basophils, smooth muscle contraction, increase of vascularpermeability, and lysis of cells (Hugh, Complement 1986, 3:111).Complement activation products also induce the generation of toxicoxygen radicals and the synthesis and release of arachidonic acidmetabolites and cytokines, in particular by phagocytes, which furtheramplifies the inflammatory response.

Although complement is an important line of defense against pathogenicorganisms, its activation can also confer damage to otherwise healthyhost cells. Inhibition of complement activation is therefore thought tobe beneficial in treating and preventing complement-mediated tissuedamage. Accordingly, there remains an urgent need in the art for noveltherapeutic agents that inhibit one or more key components of thecomplement cascade.

SUMMARY OF THE INVENTION

Provided are novel monoclonal anti-human C2b antibodies andantigen-binding fragments thereof with improved features over existingantibodies. A feature of the novel antibodies is the deletion of aglycosylation site in framework region 3 (FR3) of the heavy chainvariable domain (VH). Notably, the novel antibodies provide improvedhomogeneity and therefore improved manufacturability, as well asunexpectedly improved functional properties, compared to existingantibodies. The improved functional properties include, for example,increased pI and enhanced potential for so-called antigen sweeping. Theantibodies and antigen-binding fragments thereof will find use in humantherapy.

An aspect of the invention is a monoclonal antibody or antigen-bindingfragment thereof that specifically binds to human complement factor C2,wherein said monoclonal antibody or fragment thereof comprises:

a VH domain comprising the amino acid sequence set forth in SEQ ID NO:1; and

a VL domain comprising the amino acid sequence set forth in SEQ ID NO:2;

wherein amino acid residues 72-74 (Kabat numbering) of the VH domainconsist of X₁X₂X₃, respectively, wherein X₂ is any amino acid, andX₁X₂X₃ is not NX₂S or NX₂T.

An aspect of the invention is a pharmaceutical composition comprisingthe monoclonal antibody or antigen-binding fragment thereof inaccordance with the invention, and a pharmaceutically acceptablecarrier.

An aspect of the invention is a nucleic acid molecule or plurality ofnucleic acid molecules encoding the monoclonal antibody orantigen-binding fragment thereof in accordance with the invention.

An aspect of the invention is a vector or plurality of vectorscomprising the nucleic acid molecule or the plurality of nucleic acidmolecules in accordance with the invention.

An aspect of the invention is a host cell comprising a nucleic acidmolecule or plurality of nucleic acid molecules encoding the monoclonalantibody or antigen-binding fragment thereof in accordance with theinvention.

An aspect of the invention is a host cell comprising a vector orplurality of vectors comprising the nucleic acid molecule or theplurality of nucleic acid molecules in accordance with the invention.

An aspect of the invention is a method of making a monoclonal antibodyor antigen-binding fragment thereof in accordance with the invention,the method comprising culturing a population of cells according to theinvention under conditions permitting expression of the monoclonalantibody or antigen-binding fragment thereof.

An aspect of the invention is a method of inhibiting activation of theclassical or lectin pathway in a subject, comprising administering to asubject in need thereof an effective amount of the monoclonal antibodyor antigen-binding fragment thereof in accordance with the invention.

The following embodiments apply to all aspects of the invention.

In certain embodiments, X₁X₂X₃ consists of DX₂S.

In certain embodiments, X₁X₂X₃ consists of DKS.

In certain embodiments, the VH domain comprises the amino acid sequenceset forth in SEQ ID NO: 3.

In certain embodiments, the VL domain comprises the amino acid sequenceset forth in SEQ ID NO: 2.

In certain embodiments, the VH domain comprises the amino acid sequenceset forth in SEQ ID NO: 3, and the VL domain comprises the amino acidsequence set forth in SEQ ID NO: 2.

In certain embodiments, the monoclonal antibody or antigen-bindingfragment thereof comprises a full-length monoclonal antibody.

In certain embodiments, the monoclonal antibody comprises a human IgGheavy chain constant domain.

In certain embodiments, the heavy chain constant domain comprises ahuman IgG1 heavy chain constant domain. In certain embodiments, thehuman IgG1 heavy chain constant domain comprises the amino acid sequenceset forth in SEQ ID NO: 4.

In certain embodiments, the heavy chain constant domain comprises ahuman IgG4 heavy chain constant domain. In some embodiments, the humanIgG4 heavy chain constant domain comprises the amino acid sequence setforth in SEQ ID NO: 5.

In certain embodiments, the monoclonal antibody comprises a heavy chaincomprising the amino acid sequence set forth as SEQ ID NO: 6 and a lightchain comprising the amino acid sequence set forth as SEQ ID NO: 7.

In certain embodiments, the monoclonal antibody comprises a heavy chaincomprising the amino acid sequence set forth as SEQ ID NO: 8 and a lightchain comprising the amino acid sequence set forth as SEQ ID NO: 7.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a polyacrylamide gel loaded with indicated samples.Larger molecular weight bands for samples in lanes 4, 5, 8, and 9(arrows) show band splitting and shifting for antibodies with VH3 andVH4.

FIG. 2 is a graph depicting total levels of indicated antibodies overthe course of 31 days in cynomolgus monkeys. The following antibodieswere tested: BRO2-glyc-IgG4 (monkeys 1 and 2, glycosylated VH) andBRO2-IgG4 (monkeys 5 and 6, non-glycosylated VH).

FIGS. 3A-3I are graphs depicting levels of free C2 (plotted as OD 450 nmover time) in serum over the course of 31 days from administration ofvarious monoclonal antibodies to cynomolgus monkeys. The followingantibodies were tested: BRO2-glyc-IgG4 (FIG. 3A; monkeys 1 and 2),negative control (FIG. 3B; monkeys 3 and 4), BRO2-IgG4 (FIG. 3C; monkeys5 and 6), BRO2-IgG4-NH (FIG. 3D; monkeys 7 and 8), BRO2-IgG1-LALA-NH(FIG. 3E; ARGX-117; monkeys 9 and 10), His1-IgG4 (FIG. 3F; monkeys 11and 12), His1-IgG4-NH (FIG. 3G; monkeys 13 and 14), His1-IgG1-LALA-NH(FIG. 3H; monkeys 15 and 16), and His2-IgG4 (FIG. 3I; monkeys 17 and18).

FIG. 4 is a graph depicting average free C2 levels (plotted as OD 450 nmover time) in serum over the course of 31 days from cynomolgus monkeysadministered various indicated monoclonal antibodies.

FIG. 5 is a graph depicting free C2 levels (plotted as OD 450 nm overtime) in serum of cynomolgus monkeys treated with indicatednon-glycosylated antibodies.

FIGS. 6A-6D are a series of graphs depicting free C2 levels (plotted asOD 450 nm) in cynomolgus monkeys as determined at indicated times priorto or following administration of antibodies. Monkeys are as in FIGS.3A-3I. FIG. 6A, pre versus pre plus 500 mg/ml BRO-2; FIG. 6B, 4 hoursversus 1 day; FIG. 6C, 4 hours versus 2 days; FIG. 6D, day 11 versus day27. ADA, anti-drug antibody.

FIGS. 7A-7P are a series of graphs depicting immunogenicity (plotted asOD 450 nm) over 30 days of anti-C2 antibodies or negative controlmonoclonal antibody administered to cynomolgus monkeys. Monkeys are asin FIGS. 3A-3I. FIG. 7A, monkey 1; FIG. 7B, monkey 2; FIG. 7C, monkey 5;FIG. 7D, monkey 6; FIG. 7E, monkey 7; FIG. 7F, monkey 8; FIG. 7G, monkey9; FIG. 7H, monkey 10; FIG. 7I, monkey 11; FIG. 7J, monkey 12; FIG. 7K,monkey 13; FIG. 7L, monkey 14; FIG. 7M, monkey 15; FIG. 7N, monkey 16;FIG. 7O, monkey 17; FIG. 7P, monkey 18.

FIGS. 8A-8F are a series of graphs depicting immunogenicity (plotted asOD 450 nm over time) over 60 days of anti-C2 monoclonal antibodiesadministered to cynomolgus monkeys. Monkeys are as in FIGS. 3A-3I. FIG.8A, monkey 5; FIG. 8B, monkey 6; FIG. 8C, monkey 9; FIG. 8D, monkey 10;FIG. 8E, monkey 15; FIG. 8F, monkey 16. ADA, anti-drug antibody.

FIGS. 9A-9D depict ARGX-117 binding to C2 assessed by Western blotanalysis and surface plasmon resonance (SPR). FIG. 9A depicts Westernblot analysis of serum with ARGX-117 (representative result): Lane 1: MWsize marker; Lane 2: recombinant human C2 control (size about 100 kDa);Lane 3: serum; Lane 4: induction of complement activation by addition ofaggregated IgG to serum and incubation at 37° C.; Lane 5: C2-deficientserum.

FIG. 9B depicts SPR analysis with C2 immobilized on chip and differentARGX-117 Fabs as eluate.

FIG. 9C depicts SPR analysis with biotin-C4b immobilized tostreptavidin-chip and human C2 with and without mAbs as eluate; black:no pre-incubation; grey: anti-FXI; control human IgG4 mAb; turquoise:non-inhibitory anti-C2 clone anti-C2-63, i.e., clone 63 recognizing thelarge subunit of C2 (C2a); red: ARGX-117; all at 5 to 1 molar ratios;curves were normalized to signal just before the injection of C2 on theC4b chips.

FIG. 9D depicts SPR analysis with biotin-C4b immobilized tostreptavidin-chip and consecutively human C2 and mAbs as eluate; black:running buffer; grey: anti-FXI; control human IgG4 mAb; turquoise:non-inhibitory anti-C2 clone anti-C2-63; red: ARGX-117; curves werenormalized just before the addition of the mAbs.

FIG. 10 depicts a schematic representation of domain swap mutantsbetween C2 (SEQ ID NO: 21) and complement Factor B (FB) (SEQ ID NO: 50).In both proteins the small fragment (C2b in complement C2; SEQ ID NO: 44or FBa in complement Factor B; SEQ ID NO: 51) consists of three Sushi(or complement control protein (CCP)) domains, whereas the largefragment is composed of a von Willebrand Factor type A (VWFA) domain anda peptidase 51 domain. Note that the sequences in between the individualdomains were not taken along in these mutants but may also consist ofepitopes. Additional sequences include C2a, SEQ ID NO: 43; C2b 51, SEQID NO: 45; C2b S2, SEQ ID NO: 46; C2b S3, SEQ ID NO: 47; C2 VWFA, SEQ IDNO: 48; C2 peptidase 51, SEQ ID NO: 49; FBb, SEQ ID NO: 52; FBa 51, SEQID NO: 53; FBa S2, SEQ ID NO: 54; FBa S3, SEQ ID NO: 55; FB VWFA, SEQ IDNO: 56; and FB peptidase 1, SEQ ID NO: 57.

FIG. 11 depicts results obtained with an anti-FLAG ELISA performed ondomain-swap mutants. Five-times diluted supernatants from transfectedHEK293 cells were used for coating, and anti-FLAG mouse monoclonal Ab incombination with HRP-labeled anti-mouse IgG were used for detection.

FIG. 12 depicts results obtained with a domain swap ELISA performed withanti-C2-5F2.4. Anti-C2-5F2.4 mAb (human IgG4 S241P VH4/VL3 LC-13/03-163ABioceros) was used for coating, plates were incubated with 20 timesdiluted supernatant of HEK293 transfectants, and binding was detected byan anti-FLAG Ab. Representative results from two independent experimentswith similar outcome.

FIG. 13 depicts an amino acid sequence alignment of human and mouseSushi 2 (S2) domain of C2b. Human S2, SEQ ID NO: 46; Mouse S2, SEQ IDNO: 58. Stars indicate sequence identity.

FIG. 14 depicts results obtained with an anti-FLAG ELISA on fine mappingmutants. Undiluted supernatants from transfected HEK293 cells were usedfor coating, and biotin-labeled anti-FLAG mouse monoclonal Ab incombination with HRP-labeled SA conjugate were used for detection.

FIG. 15 depicts results on fine mapping mutants. Anti-C2-5F2.4 mAb(human IgG4 S241P VH4/VL3 LC-13/03-163A Bioceros) was used for coating,plates were incubated with 20 times diluted supernatant of HEK293transfectants, and binding was detected by an anti-FLAG Ab.

FIG. 16 depicts a plan of cluster mapping mutants using three amino acidmutations for each cluster, locations for which indicated with bold fontin the human sequence. Each human sequence was mutated to substitute thecorresponding mouse amino acid for the human amino acid shown in bold.Human S2, SEQ ID NO: 46; Mouse S2, SEQ ID NO: 58. Stars indicatesequence identity.

FIGS. 17A and 17B depict anti-FLAG ELISA on cluster mapping mutants.FIG. 17A depicts five-times diluted supernatants from transfected HEK293cells were used for coating and anti-FLAG mouse monoclonal Ab incombination with HRP-labeled anti-mouse IgG as detection. GFP, greenfluorescent protein.

FIG. 17B depicts anti-C2-5F2.4 binding to cluster mutants. Anti-C2-5F2.4mAb (human IgG4 S241P VH4/VL3, LC-13/03-163A, Bioceros) was used ascoat, plates were incubated with 20-times diluted supernatant of HEK293transfectants, and binding was detected by an anti-FLAG Ab. GFP, greenfluorescent protein.

DETAILED DESCRIPTION Definitions

“Antibody” or “Immunoglobulin”—As used herein, the term “immunoglobulin”includes a polypeptide having a combination of two heavy and two lightchains whether or not it possesses any relevant specificimmunoreactivity. As used herein, the term “antibody” refers to suchassemblies which have significant specific immunoreactive activity to anantigen of interest (e.g. the complex of complement proteins includingC2). The term “C2 antibodies” is used herein to refer to antibodieswhich exhibit immunological specificity for the complex of complementproteins including C2, particularly the human C2 protein and the domainswhich are formed through cleavage of C2, and in some cases specieshomologues thereof. Antibodies and immunoglobulins comprise light andheavy chains, with or without an interchain covalent linkage betweenthem. Basic immunoglobulin structures in vertebrate systems arerelatively well understood.

Five distinct classes of antibody (IgG, IgM, IgA, IgD, and IgE) can bedistinguished biochemically. All five classes of antibodies are withinthe scope of the present invention. The following discussion willgenerally be directed to the IgG class of immunoglobulin molecules. Withregard to IgG, immunoglobulins typically comprise two identical lightpolypeptide chains of molecular weight approximately 23,000 Daltons, andtwo identical heavy chains of molecular weight 53,000-70,000. The fourchains are joined by disulfide bonds in a “Y” configuration wherein thelight chains bracket the heavy chains starting at the mouth of the “Y”and continuing through the variable region.

The light chains of an antibody are classified as either kappa (κ) orlambda (λ). Each heavy chain class may be bound with either a kappa orlambda light chain. In general, the light and heavy chains arecovalently bonded to each other, and the “tail” portions of the twoheavy chains are bonded to each other by covalent disulfide linkages ornon-covalent linkages when the immunoglobulins are generated either byhybridomas, B cells or genetically engineered host cells. In the heavychain, the amino acid sequences run from an N-terminus at the forkedends of the Y configuration to the C-terminus at the bottom of eachchain. Those skilled in the art will appreciate that heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, or ε)with some subclasses among them (e.g., γ1-γ4). It is the nature of thischain that determines the “class” of the antibody as IgG, IgM, IgA, IgDor IgE, respectively. The immunoglobulin subclasses (isotypes) e.g.,IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are knownto confer functional specialization. Modified versions of each of theseclasses and isotypes are readily discernible to the skilled artisan inview of the instant disclosure and, accordingly, are within the scope ofthe instant invention.

As indicated above, the variable region of an antibody allows theantibody to selectively recognize and specifically bind epitopes onantigens. That is, the VL domain and VH domain of an antibody combine toform a variable region that defines a three-dimensional antigen-bindingsite. This quaternary antibody structure forms the antigen-binding sitepresent at the end of each arm of the Y. More specifically, theantigen-binding site is defined by three complementary determiningregions (CDRs) on each of the VH and VL chains.

“Binding Molecule”—As used herein, the term “binding molecule” is ageneric term intended to encompass the antibodies and antigen-bindingfragments thereof in accordance with the present disclosure.

“Binding Site”—As used herein, the term “binding site” comprises aregion of a polypeptide which is responsible for selectively binding toa target antigen of interest. Binding domains comprise at least onebinding site. Exemplary binding domains include an antibody variabledomain. The antibody molecules of the invention may comprise a singlebinding site or multiple (e.g., two, three or four) binding sites.

“Variable region” or “variable domain”—The term “variable” refers to thefact that certain portions of the variable domains VH and VL differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its target antigen. However,the variability is not evenly distributed throughout the variabledomains of antibodies. It is concentrated in three segments called“hypervariable loops” in each of the VL domain and the VH domain whichform part of the antigen-binding site. The first, second and thirdhypervariable loops of the Vlambda light chain domain are referred toherein as L1(λ), L2(λ) and L3(λ) and may be defined as comprisingresidues 24-33 (L1(λ), consisting of 9, 10 or 11 amino acid residues),49-53 (L2(λ), consisting of 3 residues) and 90-96 (L3(λ), consisting of5 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)).The first, second and third hypervariable loops of the Vkappa lightchain domain are referred to herein as L1(κ), L2(κ) and L3(κ) and may bedefined as comprising residues 25-33 (L1(κ), consisting of 6, 7, 8, 11,12 or 13 residues), 49-53 (L2(κ), consisting of 3 residues) and 90-97(L3(κ), consisting of 6 residues) in the VL domain (Morea et al.,Methods 20:267-279 (2000)). The first, second and third hypervariableloops of the VH domain are referred to herein as H1, H2 and H3 and maybe defined as comprising residues 25-33 (H1, consisting of 7, 8 or 9residues), 52-56 (H2, consisting of 3 or 4 residues) and 91-105 (H3,highly variable in length) in the VH domain (Morea et al., Methods20:267-279 (2000)).

Unless otherwise indicated, the terms L1, L2 and L3 respectively referto the first, second and third hypervariable loops of a VL domain, andencompass hypervariable loops obtained from both Vkappa and Vlambdaisotypes. The terms H1, H2 and H3 respectively refer to the first,second and third hypervariable loops of the VH domain, and encompasshypervariable loops obtained from any of the known heavy chain isotypes,including γ, μ, α, δ or ε.

The hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise partof a “complementarity determining region” or “CDR”, as defined below.The terms “hypervariable loop” and “complementarity determining region”are not strictly synonymous, since the hypervariable loops (HVs) aredefined on the basis of structure, whereas complementarity determiningregions (CDRs) are defined based on sequence variability (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1983) and thelimits of the HVs and the CDRs may be different in some VH and VLdomains.

The CDRs of the VL and VH domains can typically be defined as comprisingthe following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2) and89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or31-35b (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in the heavy chainvariable domain; (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). Thus, the HVs may be comprised within thecorresponding CDRs and references herein to the “hypervariable loops” ofVH and VL domains should be interpreted as also encompassing thecorresponding CDRs, and vice versa, unless otherwise indicated.

The more highly conserved portions of variable domains are called theframework region (FR), as defined below. The variable domains of nativeheavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4,respectively), largely adopting a β-sheet configuration, connected bythe three hypervariable loops. The hypervariable loops in each chain areheld together in close proximity by the FRs and, with the hypervariableloops from the other chain, contribute to the formation of theantigen-binding site of antibodies. Structural analysis of antibodiesrevealed the relationship between the sequence and the shape of thebinding site formed by the complementarity determining regions (Chothiaet al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol.Biol, 215:175-182 (1990)). Despite their high sequence variability, fiveof the six loops adopt just a small repertoire of main-chainconformations, called “canonical structures”. These conformations arefirst of all determined by the length of the loops and secondly by thepresence of key residues at certain positions in the loops and in theframework regions that determine the conformation through their packing,hydrogen bonding or the ability to assume unusual main-chainconformations.

“Framework region”—The term “framework region” or “FR region” as usedherein, includes the amino acid residues that are part of the variableregion, but are not part of the CDRs (e.g., using the Kabat definitionof CDRs). Therefore, a variable region framework is between about100-120 amino acids in length but includes only those amino acidsoutside of the CDRs. For the specific example of a heavy chain variabledomain and for the CDRs as defined by Kabat et al., framework region 1corresponds to the domain of the variable region encompassing aminoacids 1-30; framework region 2 corresponds to the domain of the variableregion encompassing amino acids 36-49; framework region 3 corresponds tothe domain of the variable region encompassing amino acids 66-94, andframework region 4 corresponds to the domain of the variable region fromamino acids 103 to the end of the variable region. The framework regionsfor the light chain are similarly separated by each of the light chainvariable region CDRs. Similarly, using the definition of CDRs by Chothiaet al. or McCallum et al. the framework region boundaries are separatedby the respective CDR termini as described above. In preferredembodiments the CDRs are as defined by Kabat.

In naturally occurring antibodies, the six CDRs present on eachmonomeric antibody are short, non-contiguous sequences of amino acidsthat are specifically positioned to form the antigen-binding site as theantibody assumes its three-dimensional configuration in an aqueousenvironment. The remainder of the heavy and light variable domains showless inter-molecular variability in amino acid sequence and are termedthe framework regions. The framework regions largely adopt a n-sheetconformation and the CDRs form loops which connect, and in some casesform part of, the n-sheet structure. Thus, these framework regions actto form a scaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. Theantigen-binding site formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope. The position of CDRs can bereadily identified by one of ordinary skill in the art.

“Non-glycosylated”—As used herein, the term “non-glycosylated” refers toa form of antibody or antigen-binding fragment thereof which lacksglycosylation at a potential glycosylation site in the antibody orantigen-binding fragment. In certain embodiments, the term“non-glycosylated” refers to a form of antibody or antigen-bindingfragment thereof which lacks glycosylation at a potential N-linkedglycosylation site in antibody or antigen-binding fragment. In certainembodiments, the term “non-glycosylated” refers to a form of antibody orantigen-binding fragment thereof which lacks glycosylation at apotential N-linked glycosylation site in the variable region of theheavy chain.

“Constant region”—As used herein, the term “constant region” refers tothe portion of the antibody molecule outside of the variable domains orvariable regions. Immunoglobulin light chains have a single domain“constant region”, typically referred to as the “CL or CL1 domain”. Thisdomain lies C-terminal to the VL domain. Immunoglobulin heavy chainsdiffer in their constant region depending on the class of immunoglobulin(γ, μ, α, δ, ε). Heavy chains γ, α and δ have a constant regionconsisting of three immunoglobulin domains (referred to as CH1, CH2 andCH3) with a flexible hinge region separating the CH1 and CH2 domains.Heavy chains μ and ε have a constant region consisting of four domains(CH1-CH4). The constant domains of the heavy chain are positionedC-terminal to the VH domain.

The numbering of the amino acids in the heavy and light immunoglobulinchains run from the N-terminus at the forked ends of the Y configurationto the C-terminus at the bottom of each chain. Different numberingschemes are used to define the constant domains of the immunoglobulinheavy and light chains. In accordance with the EU numbering scheme, theheavy chain constant domains of an IgG molecule are identified asfollows: CH1—amino acid residues 118-215; CH2—amino acid residues231-340; CH3—amino acid residues 341-446. The “hinge region” includesthe portion of a heavy chain molecule that joins the CH1 domain to theCH2 domain. This hinge region comprises approximately 25 residues and isflexible, thus allowing the two N-terminal antigen-binding regions tomove independently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux K. H. et al. J.Immunol. 161:4083-90 1998). Antibodies of the invention comprising a“fully human” hinge region may contain one of the hinge region sequencesshown in Table 1 below.

TABLE 1 Human hinge sequenes Upper Middle Lower IgG hinge hinge hingeIgG1 EPKSCDKTHT CPPCP APELLGGP (SEQ ID (SEQ ID (SEQ ID NO: 9) NO: 10)NO: 11) IgG2 ERK CCVECPPPCP APPVAGP (SEQ ID (SEQ ID (SEQ ID NO: 12)NO: 13) NO: 14) IgG3 ELKTPLG CPRCP(EPKSCDT APELLGGP DTTHT PPPCPRCP)₃(SEQ ID (SEQ ID (SEQ ID NO: 17) NO: 15) NO: 16) IgG4 ESKYGPP CPSCPAPEFLGGP (SEQ ID (SEQ ID (SEQ ID NO: 18) NO: 19) NO: 20)

“Fragment”—The term “fragment”, as used in the context of antibodies ofthe invention, refers to a part or portion of an antibody or antibodychain comprising fewer amino acid residues than an intact or completeantibody or antibody chain. The term “antigen-binding fragment” refersto a polypeptide fragment of an immunoglobulin or antibody thatspecifically binds antigen or competes with intact antibody (i.e., withthe intact antibody from which they were derived) for antigen-specificbinding (e.g., specific binding to the C2 protein or to a portionthereof). As used herein, the term “fragment” of an antibody moleculeincludes antigen-binding fragments of antibodies, for example, anantibody light chain variable domain (VL), an antibody heavy chainvariable domain (VH), a single chain antibody (scFv), a F(ab′)2fragment, a Fab fragment, an Fd fragment, an Fv fragment, a one-armed(monovalent) antibody, diabodies, triabodies, tetrabodies or anyantigen-binding molecule formed by combination, assembly or conjugationof such antigen-binding fragments. The term “antigen-binding fragment”as used herein is further intended to encompass antibody fragmentsselected from the group consisting of unibodies, domain antibodies andnanobodies. Fragments can be obtained, e.g., via chemical or enzymatictreatment of an intact or complete antibody or antibody chain, or byrecombinant means.

Complement Component C2

The second component of human complement (C2) is a 90-100 kDaglycoprotein which participates in the classical and lectin pathways ofcomplement activation. C2 can be activated by C1s of the classicalpathway or by activated MASP2 of the lectin pathway. C2 binds tosurface-bound C4b (in the presence of Mg′) to form a C4bC2 complex,which then is cleaved by activated C1s or MASP2 into two fragments: alarger 70 kDa fragment, traditionally designated C2a, which remainsattached to C4b to form a C3-convertase C4bC2a, and a smaller 30 kDaN-terminal fragment, traditionally designated C2b, which is releasedinto the fluid phase. Some authors have recently reversed designationsof C2a and C2b, such that C2b refers to the bigger 70 kDa fragment, andC2a refers to the smaller 30 kDa fragment. As used herein, C2a shallrefer to the bigger 70 kDa fragment, and C2b shall refer to the smaller30 kDa fragment. Once activated and bound to C4b, C2a constitutes thecatalytic subunit of the C3 and C5 convertases which are able to cleaveC3 and C5, respectively.

The amino acid sequence of human C2 is known (GenBank Accession No.NM_000063) and shown as SEQ ID NO: 21.

Amino Acid Sequence of human C2 (SEQ ID NO: 21):MGPLMVLFCLLFLYPGLADSAPSCPQNVNISGGTFTLSHGWAPGSLLTYSCPQGLYPSPASRLCKSSGQWQTPGATRSLSKAVCKPVRCPAPVSFENGIYTPRLGSYPVGGNVSFECEDGFILRGSPVRQCRPNGMWDGETAVCDNGAGHCPNPGISLGAVRTGFRFGHGDKVRYRCSSNLVLTGSSERECQGNGVWSGTEPICRQPYSYDFPEDVAPALGTSFSHMLGATNPTQKTKESLGRKIQIQRSGHLNLYLLLDCSQSVSENDFLIFKESASLMVDRIFSFEINVSVAIITFASEPKVLMSVLNDNSRDMTEVISSLENANYKDHENGTGTNTYAALNSVYLMMNNQMRLLGMETMAWQEIRHAIILLTDGKSNMGGSPKTAVDHIREILNINQKRNDYLDIYAIGVGKLDVDWRELNELGSKKDGERHAFILQDTKALHQVFEHMLDVSKLTDTICGVGNMSANASDQERTPWHVTIKPKSQETCRGALISDQWVLTAAHCFRDGNDHSLWRVNVGDPKSQWGKEFLIEKAVISPGFDVFAKKNQGILEFYGDDIALLKLAQKVKMSTHARPICLPCTMEANLALRRPQGSTCRDHENELLNKQSVPAHFVALNGSKLNINLKMGVEWTSCAEVVSQEKTMFPNLTDVREVVTDQFLCSGTQEDESPCKGESGGAVFLERRFRFFQVGLVSWGLYNPCLGSADKNSRKRAPRSKVPPPRDFHINLFRMQPWLRQHLGDVLNFLPL

As with many other plasma proteins, C2 has a modular structure. Startingfrom its N-terminus, C2 consists of three complement control proteinmodules (CCP1-3, also known as short consensus repeats (SCR) orsushi-domain repeats), a von Willebrand factor type A (vWFA) domaincontaining a metal-ion-dependent adhesion site, and a serine protease(SP) domain (Arlaud et al., Adv Immunol 1998, 69: 249). Electronmicroscopy studies have revealed that C2 consists of three domains. Thethree CCP modules (CCP1-3) together form the N-terminal domain, whichcorresponds to C2b. The vWFA domain constitutes the second domain, andthe SP domain makes up the third domain. The second and third domainstogether constitute the larger C2a portion of the molecule.

CCP modules are common structural motifs that occur in a number ofproteins. These globular units consist of approximately 60 amino acidresidues and are folded into a compact six- to eight-stranded β-sheetstructure built around four invariant disulfide-bonded cysteine residues(Norman et al., J Mol Biol 1991, 219: 717). Neighboring CCP modules arecovalently attached by poorly conserved linkers.

The initial binding of C2 to surface-bound C4b is mediated by twolow-affinity sites, one on C2b (Xu & Volanakis, J Immunol 1997, 158:5958) and the other on the vWFA domain of C2a (Horiuchi et al., JImmunol 1991, 47: 584). Though the crystal structure of C2b and C2a havebeen determined to 1.8 Å resolution (Milder et al., Structure 2006, 14:1587; Krishnan et al., J Mol Biol 2007, 367: 224; Krishnan et al., ActaCristallogr D Biol Crystallogr 2009, D65: 266), the exact topology andstructure of the amino acid residues constituting the contact site(s)for C4 and C3 on C2 are unknown. Thus the amino acid residues of C2involved in the interaction with C4 remain to be established (Krishnanet al., Acta Cristallogr D Biol. Crystallogr 2009, D65: 266).

Anti-C2 Antibodies

An aspect of the invention is a monoclonal antibody or antigen-bindingfragment thereof that specifically binds to human complement factor C2,wherein said monoclonal antibody or fragment thereof comprises:

a VH domain comprising the amino acid sequence set forth in SEQ ID NO:1; and

a VL domain comprising the amino acid sequence set forth in SEQ ID NO:2;

wherein amino acid residues 72-74 (Kabat numbering) of the VH domainconsist of X₁X₂X₃, respectively, wherein X₂ is any amino acid, andX₁X₂X₃ is not NX₂S or NX₂T.

The VH domain comprises complementarity determining regions (CDRs)HCDR1, HCDR2, and HCDR3. The VL domain comprises CDRs LCDR1, LCDR2, andLCDR3. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,and LCDR3 are shown in Table 2.

TABLE 2 CDRs HCDR1 DYNMD (SEQ ID NO: 22) HCDR2 DINPNYESTGYNQKFKG(SEQ ID NO: 23) HCDR3 EDDHDAFAY (SEQ ID NO: 24) LCDR1 RASKSVRTSGYNYMH(SEQ ID NO: 25) LCDR2 LASNLKS (SEQ ID NO: 26) LCDR3 QHSRELPYT(SEQ ID NO: 27)

In certain embodiments, the monoclonal antibody or antigen-bindingfragment thereof specifically binds to human complement factor C2b. Incertain embodiments, the monoclonal antibody or antigen-binding fragmentthereof specifically binds to an epitope in a portion of humancomplement factor C2 corresponding to human complement factor C2b.

In certain embodiments, the variable domain of the heavy chain isnon-glycosylated. In certain embodiments the amino acid sequence of thevariable domain of the heavy chain does not include a potentialglycosylation site which is characterized by the sequence N—X—S/T, whereN represents asparagine, X represents any amino acid, and S/T representsserine or threonine. Accordingly, in certain embodiments, antibodieswith a VH domain comprising the sequence N—X—S/T can be modified so thatthese residues consist of X₁X₂X₃, respectively, wherein X₂ is any aminoacid, and X₁X₂X₃ is not NX₂S or NX₂T. That is, X₁ can be any amino acidother than N, and/or X₃ can be any amino acid other than S or T. Incertain embodiments, antibodies with a VH domain comprising the sequenceN—X—S or N—X-T can be modified so that these three residues consist ofD-X—S, respectively. In certain other embodiments, antibodies with a VHdomain comprising the sequence N—X—S or N—X-T can be modified so thatthese three residues consist of D-X-T, respectively.

In certain embodiments, heavy chain amino acids at residues 72-74 (Kabatnumbering) consist of X₁X₂X₃, respectively, wherein X₂ is any aminoacid, and X₁X₂X₃ is not NX₂S or NX₂T.

In certain embodiments, heavy chain amino acids at residues 72-74 (Kabatnumbering) consist of DX₂S.

In certain embodiments, heavy chain amino acids at residues 72-74 (Kabatnumbering) consist of DKS.

In certain embodiments, the VH domain comprises the amino acid sequenceset forth in SEQ ID NO: 3.

In certain embodiments, the amino acid sequence of the VH domainconsists of the sequence set forth in SEQ ID NO: 3.

In certain embodiments, the VL domain comprises the amino acid sequenceset forth in SEQ ID NO: 2.

In certain embodiments, the amino acid sequence of the VL domainconsists of the sequence set forth in SEQ ID NO: 2.

In certain embodiments, the VH domain comprises the amino acid sequenceset forth in SEQ ID NO: 3, and the VL domain comprises the amino acidsequence set forth in SEQ ID NO: 2.

In certain embodiments, the amino acid sequence of the VH domainconsists of the sequence set forth in SEQ ID NO: 3, and the amino acidsequence of the VL domain consists of the sequence set forth in SEQ IDNO: 2.

The amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 2 are shown inTable 3. SEQ ID NO: 2 corresponds to the VL (VK3) domain of humanized5F2.4 (BRO2) disclosed in U.S. Pat. No. 9,944,717 to Broteio Pharma B.V.Also shown in Table 3, SEQ ID NO: 28 corresponds to the VH (VH4) domainof humanized 5F2.4 (BRO2) disclosed in U.S. Pat. No. 9,944,717 which isincorporated by reference herein.

TABLE 3 VH and VL Domains SEQ ID ID Sequence NO: 5F2.4EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVR 28 VH4QATGQGLEWIGDINPNYESTGYNQKFKGRATMTVNKSISTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLV TVSS VH4.2EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVR  1 genericQATGQGLEWIGDINPNYESTGYNQKFKGRATMTVX₁X₂X₃ISTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQG TLVTVSS VH4.2EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVR  3 ARGX-QATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSI 117STAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLV TVSS 5F2.4DNVLTQSPDSLAVSLGERATISCRASKSVRTSGYNYMH  2 VK3WYQQKPGQPPKLLIYLASNLKSGVPDRFSGSGSGTDFTLTISSLQAEDAATYYCQHSRELPYTFGQGTKLEIK

In certain embodiments, the monoclonal antibodies of the inventioninclude the CH1 domain, hinge domain, CH2 domain, and CH3 domain of ahuman antibody, in particular human IgG1, IgG2, IgG3 or IgG4.

In certain embodiments, the antibody includes the CH1 domain, hingedomain, CH2 domain, and CH3 domain of a human IgG1 and includes thesubstitutions L234A and L235A in the CH2 domain.

In certain embodiments, the antibody includes the CH1 domain, hingedomain, CH2 domain, and CH3 domain of a human IgG1 and includes thesubstitutions H433K and N434F in the CH3 domain.

In certain embodiments, the antibody includes the CH1 domain, hingedomain, CH2 domain, and CH3 domain of a human IgG1 and includes thesubstitutions L234A and L235A in the CH2 domain, and the substitutionsH433K and N434F in the CH3 domain.

In certain embodiments, the antibody includes the CH1 domain, hingedomain, CH2 domain, and CH3 domain of a human IgG4. In certainembodiments, the antibody includes the CH1 domain, hinge domain, CH2domain, and CH3 domain of a human IgG4 and includes the substitutionS228P in the hinge domain.

In certain embodiments, the antibody includes the CH1 domain, hingedomain, CH2 domain, and CH3 domain of a human IgG4 and includes thesubstitution L445P in the CH3 domain.

In certain embodiments, the antibody includes the CH1 domain, hingedomain, CH2 domain, and CH3 domain of a human IgG4 and includes both thesubstitution S228P in the hinge domain and the substitution L445P in theCH3 domain.

In certain embodiments, the antibody includes the CH1 domain, hingedomain, CH2 domain, and CH3 domain of a human IgG4 and includes thesubstitutions H433K and N434F in the CH3 domain.

In certain embodiments, the antibody includes the CH1 domain, hingedomain, CH2 domain, and CH3 domain of a human IgG4 and includes thesubstitution S228P in the hinge domain, and the substitutions H433K andN434F in the CH3 domain.

In certain embodiments, the antibody includes the CH1 domain, hingedomain, CH2 domain, and CH3 domain of a human IgG4 and includes thesubstitutions H433K, N434F, and L445P in the CH3 domain.

In certain embodiments, the antibody includes the CH1 domain, hingedomain, CH2 domain, and CH3 domain of a human IgG4 and includes thesubstitution S228P in the hinge domain, and the substitutions H433K,N434F, and L445P in the CH3 domain.

In certain embodiments, the monoclonal antibody comprises a human IgGheavy chain constant domain. In certain embodiments, the heavy chainconstant domain comprises a human IgG1 heavy chain constant domain. Incertain embodiments, the heavy chain constant domain consists of a humanIgG1 heavy chain constant domain.

In certain embodiments, the heavy chain constant domain comprises ahuman IgG1 heavy chain constant domain comprising the amino acidsequence set forth as SEQ ID NO: 29. In certain embodiments, the aminoacid sequence of the heavy chain constant domain consists of thesequence set forth as SEQ ID NO: 29.

In certain embodiments, the heavy chain constant domain comprises ahuman IgG1 heavy chain constant domain comprising the amino acidsequence set forth as SEQ ID NO: 4. In certain embodiments, the aminoacid sequence of the heavy chain constant domain consists of thesequence set forth as SEQ ID NO: 4.

In certain embodiments, the heavy chain constant domain comprises ahuman IgG4 heavy chain constant domain. In certain embodiments, theheavy chain constant domain consists of a human IgG4 heavy chainconstant domain.

In certain embodiments, the heavy chain constant domain comprises ahuman IgG4 heavy chain constant domain comprising the amino acidsequence set forth as SEQ ID NO: 30. In certain embodiments, the aminoacid sequence of the heavy chain constant domain consists of thesequence set forth as SEQ ID NO: 30.

In certain embodiments, the heavy chain constant domain comprises ahuman IgG4 heavy chain constant domain comprising the amino acidsequence set forth as SEQ ID NO: 31. In certain embodiments, the aminoacid sequence of the heavy chain constant domain consists of thesequence set forth as SEQ ID NO: 31.

In certain embodiments, the heavy chain constant domain comprises ahuman IgG4 heavy chain constant domain comprising the amino acidsequence set forth as SEQ ID NO: 5. In certain embodiments, the aminoacid sequence of the heavy chain constant domain consists of thesequence set forth as SEQ ID NO: 5.

The amino acid sequences of SEQ ID NOs: 4, 5, and 29-31 are shown inTable 4.

TABLE 4 Heavy Chain Constant Domains SEQ ID ID Sequence NO: HumanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT 29 IgG1VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL (UniProt)GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK HumanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT  4 IgG1VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL LALAGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA NHancePEAAGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHE (ARGX-DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 117)VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGN VFSCSVMHEALKFHYTQKSLSLSPG HumanASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT 30 IgG4VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL (UniProt)GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGK HumanASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT 31 IgG4VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL S228PGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF L445PLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSPGK HumanASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT  5 IgG4VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL S228PGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF NHanceLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPE L445PVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFS CSVMHEALKFHYTQKSLSLSPGK

In certain embodiments, the monoclonal antibody or antigen-bindingfragment thereof comprises a full-length monoclonal antibody.

In certain embodiments, the monoclonal antibody or antigen-bindingfragment thereof consists of a full-length monoclonal antibody.

In certain embodiments, provided herein are monoclonal antibodiescomprising a heavy chain with at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% sequence identity to the amino acidsequence shown as SEQ ID NO: 32. In certain embodiments, provided hereinare monoclonal antibodies comprising a heavy chain with 100% sequenceidentity to the amino acid sequence shown as SEQ ID NO: 32. In certainembodiments, provided herein are monoclonal antibodies comprising alight chain with at least 90%, at least 95%, at least 97%, at least 98%,or at least 99% sequence identity to the amino acid sequence shown asSEQ ID NO: 7. In certain embodiments, provided herein are monoclonalantibodies comprising a light chain with at 100% sequence identity tothe amino acid sequence shown as SEQ ID NO: 7. In certain embodiments,provided herein are monoclonal antibodies comprising a heavy chain withat least 90%, at least 95%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence shown as SEQ ID NO: 32, anda light chain with at least 90%, at least 95%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence shownas SEQ ID NO: 7. In certain embodiments, provided herein are monoclonalantibodies comprising a heavy chain with 100% sequence identity to theamino acid sequence shown as SEQ ID NO: 32, and a light chain with 100%sequence identity to the amino acid sequence shown as SEQ ID NO: 7.

In certain embodiments, provided herein are monoclonal antibodiescomprising a heavy chain with at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% sequence identity to the amino acidsequence shown as SEQ ID NO: 6. In certain embodiments, provided hereinare monoclonal antibodies comprising a heavy chain with 100% sequenceidentity to the amino acid sequence shown as SEQ ID NO: 6. In certainembodiments, provided herein are monoclonal antibodies comprising alight chain with at least 90%, at least 95%, at least 97%, at least 98%,or at least 99% sequence identity to the amino acid sequence shown asSEQ ID NO: 7. In certain embodiments, provided herein are monoclonalantibodies comprising a light chain with at 100% sequence identity tothe amino acid sequence shown as SEQ ID NO: 7. In certain embodiments,provided herein are monoclonal antibodies comprising a heavy chain withat least 90%, at least 95%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence shown as SEQ ID NO: 6, anda light chain with at least 90%, at least 95%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence shownas SEQ ID NO: 7. In certain embodiments, provided herein are monoclonalantibodies comprising a heavy chain with 100% sequence identity to theamino acid sequence shown as SEQ ID NO: 6, and a light chain with 100%sequence identity to the amino acid sequence shown as SEQ ID NO: 7.

In certain embodiments, provided herein are monoclonal antibodiescomprising a heavy chain with at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% sequence identity to the amino acidsequence shown as SEQ ID NO: 33. In certain embodiments, provided hereinare monoclonal antibodies comprising a heavy chain with 100% sequenceidentity to the amino acid sequence shown as SEQ ID NO: 33. In certainembodiments, provided herein are monoclonal antibodies comprising aheavy chain with at least 90%, at least 95%, at least 97%, at least 98%,or at least 99% sequence identity to the amino acid sequence shown asSEQ ID NO: 33, and a light chain with at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence shown as SEQ ID NO: 7. In certain embodiments, providedherein are monoclonal antibodies comprising a heavy chain with 100%sequence identity to the amino acid sequence shown as SEQ ID NO: 33, anda light chain with 100% sequence identity to the amino acid sequenceshown as SEQ ID NO: 7.

In certain embodiments, provided herein are monoclonal antibodiescomprising a heavy chain with at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% sequence identity to the amino acidsequence shown as SEQ ID NO: 34. In certain embodiments, provided hereinare monoclonal antibodies comprising a heavy chain with 100% sequenceidentity to the amino acid sequence shown as SEQ ID NO: 34. In certainembodiments, provided herein are monoclonal antibodies comprising aheavy chain with at least 90%, at least 95%, at least 97%, at least 98%,or at least 99% sequence identity to the amino acid sequence shown asSEQ ID NO: 34, and a light chain with at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence shown as SEQ ID NO: 7. In certain embodiments, providedherein are monoclonal antibodies comprising a heavy chain with 100%sequence identity to the amino acid sequence shown as SEQ ID NO: 34, anda light chain with 100% sequence identity to the amino acid sequenceshown as SEQ ID NO: 7.

In certain embodiments, provided herein are monoclonal antibodiescomprising a heavy chain with at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% sequence identity to the amino acidsequence shown as SEQ ID NO: 8. In certain embodiments, provided hereinare monoclonal antibodies comprising a heavy chain with 100% sequenceidentity to the amino acid sequence shown as SEQ ID NO: 8. In certainembodiments, provided herein are monoclonal antibodies comprising aheavy chain with at least 90%, at least 95%, at least 97%, at least 98%,or at least 99% sequence identity to the amino acid sequence shown asSEQ ID NO: 8, and a light chain with at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence shown as SEQ ID NO: 7. In certain embodiments, providedherein are monoclonal antibodies comprising a heavy chain with 100%sequence identity to the amino acid sequence shown as SEQ ID NO: 8, anda light chain with 100% sequence identity to the amino acid sequenceshown as SEQ ID NO: 7.

The amino acid sequences of SEQ ID NOs: 6-8 and 32-34 are shown in Table5.

TABLE 5 Heavy Chains and Light Chains SEQ ID ID Sequence NO: HumanEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVR 32 IgG1QATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSI (UniProt)STAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVR  6 IgG1QATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSI LALASTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLV NHanceTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP (ARGX-EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP 117)SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALKFHYTQKSLSLSPGHuman EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVR 33 IgG4QATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSI (UniProt)STAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK HumanEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVR 34 IgG4QATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSI S228PSTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLV L445PTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSPGK HumanEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVR  8 IgG4QATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSI S228PSTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLV NHanceTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP L445PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALKFHYTQKSLSLSPGK LightDNVLTQSPDSLAVSLGERATISCRASKSVRTSGYNYMH  7 ChainWYQQKPGQPPKLLIYLASNLKSGVPDRFSGSGSGTDFT (ARGX-LTISSLQAEDAATYYCQHSRELPYTEGQGTKLEIKRTV 117)AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC

For embodiments wherein the heavy and/or light chains of the antibodiesare defined by a particular percentage sequence identity to a referencesequence, the heavy chain and/or light chain may retain identical CDRsequences to those present in the reference sequence such that thevariation is present only outside the CDR regions.

Unless otherwise stated in the present application, % sequence identitybetween two amino acid sequences may be determined by comparing thesetwo sequences aligned in an optimum manner and in which the amino acidsequence to be compared can comprise additions or deletions with respectto the reference sequence for an optimum alignment between these twosequences. The percentage of identity is calculated by determining thenumber of identical positions for which the amino acid residue isidentical between the two sequences, by dividing this number ofidentical positions by the total number of positions in the comparisonwindow and by multiplying the result obtained by 100 in order to obtainthe percentage of identity between these two sequences. For example, itis possible to use the BLAST program, “BLAST 2 sequences” (Tatusova etal, “Blast 2 sequences—a new tool for comparing protein and nucleotidesequences”, FEMS Microbiol Lett. 174:247-250), the parameters used beingthose given by default (in particular for the parameters “open gappenalty”: 5, and “extension gap penalty”: 2; the matrix chosen being,for example, the matrix “BLOSUM 62” proposed by the program), thepercentage of identity between the two sequences to be compared beingcalculated directly by the program.

In non-limiting embodiments, the antibodies of the present invention maycomprise CH1 domains and/or CL domains (from the heavy chain and lightchain, respectively), the amino acid sequence of which is fully orsubstantially human. Where the antibody or antigen-binding fragment ofthe invention is an antibody intended for human therapeutic use, it istypical for the entire constant region of the antibody, or at least apart thereof, to have fully or substantially human amino acid sequence.Therefore, one or more or any combination of the CL domain, CH1 domain,hinge region, CH2 domain, CH3 domain and CH4 domain (if present) may befully or substantially human with respect to its amino acid sequence.

Advantageously, the CL domain, CH1 domain, hinge region, CH2 domain, CH3domain and CH4 domain (if present) may all have fully or substantiallyhuman amino acid sequence. In the context of the constant region of ahumanized or chimeric antibody, or an antibody fragment, the term“substantially human” refers to an amino acid sequence identity of atleast 90%, or at least 92%, or at least 95%, or at least 97%, or atleast 99% with a human constant region. The term “human amino acidsequence” in this context refers to an amino acid sequence which isencoded by a human immunoglobulin gene, which includes germline,rearranged and somatically mutated genes. The invention alsocontemplates polypeptides comprising constant domains of “human”sequence which have been altered, by one or more amino acid additions,deletions or substitutions with respect to the human sequence, exceptingthose embodiments where the presence of a “fully human” hinge region isexpressly required.

The presence of a “fully human” hinge region in the C2-bindingantibodies of the invention may be beneficial both to minimizeimmunogenicity and to optimize stability of the antibody.

The C2 binding antibodies may be modified within the Fc region toincrease binding affinity for the neonatal Fc receptor FcRn. Theincreased binding affinity may be measurable at acidic pH (for examplefrom about approximately pH 5.5 to approximately pH 6.0). The increasedbinding affinity may also be measurable at neutral pH (for example fromapproximately pH 6.9 to approximately pH 7.4). In this embodiment, by“increased binding affinity” is meant increased binding affinity to FcRnrelative to binding affinity of unmodified Fc region. Typically theunmodified Fc region will possess the wild-type amino acid sequence ofhuman IgG1, IgG2, IgG3 or IgG4. In such embodiments, the increasedbinding affinity to FcRn of the antibody molecule having the modified Fcregion will be measured relative to the binding affinity of wild-typeIgG1, IgG2, IgG3 or IgG4 for FcRn.

The C2 binding antibodies may be modified within the Fc region toincrease binding affinity for the human neonatal Fc receptor FcRn. Theincreased binding affinity may be measurable at acidic pH (for examplefrom about approximately pH 5.5 to approximately pH 6.0). The increasedbinding affinity may also be measurable at neutral pH (for example fromapproximately pH 6.9 to approximately pH 7.4). In this embodiment, by“increased binding affinity” is meant increased binding affinity tohuman FcRn relative to binding affinity of unmodified Fc region.Typically the unmodified Fc region will possess the wild-type amino acidsequence of human IgG1, IgG2, IgG3 or IgG4. In such embodiments, theincreased binding affinity to human FcRn of the antibody molecule havingthe modified Fc region will be measured relative to the binding affinityof wild-type IgG1, IgG2, IgG3 or IgG4 for human FcRn.

Pharmaceutical Compositions

An aspect of the invention is a pharmaceutical composition comprising amonoclonal antibody or antigen-binding fragment thereof thatspecifically binds to human complement factor C2, and a pharmaceuticallyacceptable carrier, wherein said monoclonal antibody or fragment thereofcomprises:

a VH domain comprising the amino acid sequence set forth in SEQ ID NO:1; and

a VL domain comprising the amino acid sequence set forth in SEQ ID NO:2;

wherein amino acid residues 72-74 (Kabat numbering) of the VH domainconsist of X₁X₂X₃, respectively, wherein X₂ is any amino acid, andX₁X₂X₃ is not NX₂S or NX₂T.

A pharmaceutical composition of the invention may be formulated withpharmaceutically acceptable carriers or diluents as well as any otherknown adjuvants and excipients in accordance with conventionaltechniques such as those disclosed in (Remington: The Science andPractice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co.,Easton, Pa., 1995).

The term “pharmaceutically acceptable carrier” relates to carriers orexcipients, which are inherently non-toxic. Examples of such excipientsare, but are not limited to, saline, Ringer's solution, dextrosesolution and Hanks' solution. Non-aqueous excipients such as fixed oilsand ethyl oleate may also be used.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, micro-emulsion, liposome, or other orderedstructure suitable to high drug concentration. Examples of suitableaqueous and non-aqueous carriers which may be employed in thepharmaceutical compositions of the invention include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

The pharmaceutical compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of presence of microorganisms may be ensured both bysterilization procedures and by the inclusion of various antibacterialand antifungal agents, for example, paraben, chlorobutanol, phenol,sorbic acid, and the like. It may also be desirable to includeisotonicity agents, such as sugars, polyalcohols such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.Pharmaceutically-acceptable antioxidants may also be included, forexample (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Sterile injectable solutions can be prepared by incorporating themonoclonal antibody in the required amount in an appropriate solventwith one or a combination of ingredients, e.g., as enumerated above, asrequired, followed by sterilization microfiltration. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients, e.g., from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The pharmaceutical composition is preferably administered parenterally,preferably by intravenous (i.v.) or subcutaneous (s.c.) injection orinfusion.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intraperitoneal, subcutaneous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, transtracheal, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Prolonged absorption of the injectable anti-C2 mAbs or fragments thereofcan be brought about by including in the composition an agent thatdelays absorption, for example, monostearate salts and gelatin.

The mAbs or fragments thereof can be prepared with carriers that willprotect the compound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for the preparation of such formulations are generally known tothose skilled in the art. See, e.g., Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978.

The pharmaceutical compositions can be administered with medical devicesknown in the art.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time, orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation.

Actual dosage levels of the mAbs or fragments thereof in thepharmaceutical compositions of the present invention may be varied so asto obtain an amount of the active ingredient which is effective toachieve the desired therapeutic response for a particular patientwithout being toxic to the patient.

In one embodiment, the binding molecules, in particular antibodies,according to the invention can be administered by infusion in a weeklydosage of from 10 to 500 mg/m², such as of from 200 to 400 mg/m². Suchadministration can be repeated, e.g., 1 to 8 times, such as 3 to 5times. The administration may be performed by continuous infusion over aperiod of from 1 to 24 hours, such as a period of from 2 to 12 hours. Insome embodiments, administration may be performed by one or more bolusinjections.

In one embodiment, the binding molecules, in particular antibodies,according to the invention can be administered by infusion in a weeklydosage of from 1 to 50 mg per kg body weight (mg/kg), such as from 5 to25 mg/kg. Such administration can be repeated, e.g., 1 to 8 times, suchas 3 to 5 times. The administration may be performed by continuousinfusion over a period of from 1 to 24 hours, such as a period of from 2to 12 hours. In some embodiments, administration may be performed by oneor more bolus injections.

In yet another embodiment, the mAbs or fragments thereof or any otherbinding molecules disclosed in this invention, can be administered asmaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

Nucleic Acid Molecules and Vectors

An aspect of the invention is a nucleic acid molecule or plurality ofnucleic acid molecules encoding the monoclonal antibody orantigen-binding fragment thereof in accordance with the invention. Incertain embodiments, a single nucleic acid molecule encodes both the VHand the VL domains of the monoclonal antibody or antigen-bindingfragment thereof in accordance with the invention. In certainembodiments, a single nucleic acid molecule encodes both the heavy chain(HC) and the light chain (LC) of the monoclonal antibody orantigen-binding fragment thereof in accordance with the invention. Incertain embodiments, a first nucleic acid molecule encodes the VHdomain, and a second nucleic acid molecule encodes the VL domain of themonoclonal antibody or antigen-binding fragment thereof in accordancewith the invention. In certain embodiments, a first nucleic acidmolecule encodes the heavy chain (HC), and a second nucleic acidmolecule encodes the light chain (LC) of the monoclonal antibody orantigen-binding fragment thereof in accordance with the invention.

In certain embodiments, a nucleic acid molecule encoding the VH domaincomprises the nucleic acid sequence set forth as SEQ ID NO: 35.

In certain embodiments, a nucleic acid molecule encoding the VL domaincomprises the nucleic acid sequence set forth as SEQ ID NO: 36.

In certain embodiments, a nucleic acid molecule encoding the HCcomprises the nucleic acid sequence set forth as SEQ ID NO: 37.

In certain embodiments, a nucleic acid molecule encoding the HCcomprises the nucleic acid sequence set forth as SEQ ID NO: 38.

In certain embodiments, a nucleic acid molecule encoding the HCcomprises the nucleic acid sequence set forth as SEQ ID NO: 39.

In certain embodiments, a nucleic acid molecule encoding the HCcomprises the nucleic acid sequence set forth as SEQ ID NO: 40.

In certain embodiments, a nucleic acid molecule encoding the HCcomprises the nucleic acid sequence set forth as SEQ ID NO: 41.

In certain embodiments, a nucleic acid molecule encoding the LC domaincomprises the nucleic acid sequence set forth as SEQ ID NO: 42.

In certain embodiments, the nucleic acid sequence of a nucleic acidmolecule encoding the VH domain consists of the sequence set forth asSEQ ID NO: 35.

In certain embodiments, the nucleic acid sequence of a nucleic acidmolecule encoding the VL domain consists of the sequence set forth asSEQ ID NO: 36.

In certain embodiments, the nucleic acid sequence of a nucleic acidmolecule encoding the HC consists of the sequence set forth as SEQ IDNO: 37.

In certain embodiments, the nucleic acid sequence of a nucleic acidmolecule encoding the HC consists of the sequence set forth as SEQ IDNO: 38.

In certain embodiments, the nucleic acid sequence of a nucleic acidmolecule encoding the HC consists of the sequence set forth as SEQ IDNO: 39.

In certain embodiments, the nucleic acid sequence of a nucleic acidmolecule encoding the HC consists of the sequence set forth as SEQ IDNO: 40.

In certain embodiments, the nucleic acid sequence of a nucleic acidmolecule encoding the HC consists of the sequence set forth as SEQ IDNO: 41.

In certain embodiments, the nucleic acid sequence of a nucleic acidmolecule encoding the LC domain consists of the sequence set forth asSEQ ID NO: 42.

The nucleic acid sequences corresponding to SEQ ID NOs: 35-42 are shownin Table 6.

TABLE 6 Nucleic Acid Sequences of VH, VL, HC, and LC SEQ ID ID SequenceNO: BRO2- gaagtgcagctggtgcagtctggcgccgaagtgaagaaacctggcgcctc 35 IgG4cgtgaaggtgtcctgcaaggcttccggctacacctttaccgactacaaca VH4.2tggactgggtgcgacaggctaccggccagggcctggaatggatcggcgacatcaaccccaactacgagtccaccggctacaaccagaagttcaagggcagagccaccatgaccgtggacaagtccatctccaccgcctacatggaactgtcctccctgcggagcgaggacaccgccgtgtactactgcgccagagaggacgaccacgacgcctttgcttattggggccagggcaccctcgtgaccgtgtc ctct BRO2 VLgacaacgtgctgacccagtcccctgactccctggctgtgtctctgggcga 36gagagccaccatctcttgccgggcctctaagtccgtgcggacctccggctacaactacatgcactggtatcagcagaagcccggccagccccccaagctgctgatctacctggcctccaacctgaagtccggcgtgcccgacagattctccggctctggctctggcaccgactttaccctgaccatcagctccctgcaggccgaggatgccgccacctactactgccagcactccagagagctgccctacacctttggccagggcaccaagctggaaatcaag BRO2-gaagttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctc 37 hIgG1 HCtgtgaaggtgtcctgcaaggcttctggctacacctttaccgactacaacatggactgggtccgacaggctaccggacagggacttgagtggatcggcgacatcaaccccaactacgagtccaccggctacaaccagaagttcaagggcagagccaccatgaccgtggacaagtccatctccaccgcctacatggaactgtccagcctgagatctgaggacaccgccgtgtactactgcgccagagaggatgatcacgacgcctttgcttattggggccagggcacactggtcaccgtgtcctctgccagtacaaaaggtccaagtgtgttccctcttgctccctcatccaagagtaccagtggaggcaccgccgctcttggctgcttggttaaggattatttcccagagcctgtcactgtttcatggaactccggcgccttgacatctggtgtgcatacctttccagccgtgctgcagtcaagtggcctctacagcctcagtagcgtggtcactgtgcccagcagctctctcggcacacaaacttatatctgtaatgtgaatcataagccttcaaataccaaggtggataagaaagtggaaccaaaatcatgtgacaagacacacacctgccctccttgtccagcccccgaactgctgggtgggcccagcgtgttcctgtttcctcctaaacccaaagacactctgatgattagtaggaccccagaagtcacttgcgtggtggttgacgtgtcacatgaagatcccgaggtcaagttcaattggtatgttgacggggtcgaagttcacaacgctaaaactaaaccaagagaggaacagtataactctacctaccgggtggtgagtgttctgactgtcctccatcaagactggctgaatggcaaagaatacaagtgtaaggtgagcaacaaagccctgcccgctcctatagagaaaacaatatccaaagccaaaggtcaacctcgcgagccacaggtgtacaccctcccaccaagccgcgatgaacttactaagaaccaagtctctcttacttgcctggttaaggggttctatccatccgacattgcagtcgagtgggagtctaatggacagcctgagaacaactacaaaaccacccctcctgttctggattctgacggatctttcttcctttattctaaactcaccgtggataaaagcaggtggcagcagggcaacgtgttcagctgttccgttatgcatgaggccctgcataaccattatacccagaagtctttgtccctcagtccaggaaag BRO2-gaagttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctc 38 hIgG1-tgtgaaggtgtcctgcaaggcttctggctacacctttaccgactacaaca LALA-NHtggactgggtccgacaggctaccggacagggacttgagtggatcggcgac HCatcaaccccaactacgagtccaccggctacaaccagaagttcaagggcagagccaccatgaccgtggacaagtccatctccaccgcctacatggaactgtccagcctgagatctgaggacaccgccgtgtactactgcgccagagaggatgatcacgacgcctttgcttattggggccagggcacactggtcaccgtgtcctctgcttctaccaagggacccagcgtgttccctctggctccttccagcaagtctacctctggcggaacagctgctctgggctgcctggtcaaggactactttcctgagcctgtgaccgtgtcttggaactctggcgctctgacatctggcgtgcacacctttccagctgtgctgcagtcctccggcctgtactctctgtcctctgtcgtgaccgtgccttccagctctctgggaacccagacctacatctgcaatgtgaaccacaagccttccaacaccaaggtggacaagaaggtggaacccaagtcctgcgacaagacccacacctgtcctccatgtcctgctccagaagctgctggcggcccttccgtgtttctgttccctccaaagcctaaggacaccctgatgatctctcggacccctgaagtgacctgcgtggtggtggatgtgtctcacgaggacccagaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgctcctatcgaaaagaccatctccaaggccaagggccagcctagggaaccccaggtttacaccttgcctccatctcgggacgagctgaccaagaaccaggtgtccctgacctgtctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtctaatggccagccagagaacaactacaagacaacccctcctgtgctggactccgacggctcattctttctgtactccaagctgacagtggataagtcccggtggcagcagggcaacgtgttctcctgttctgtgatgcacgaggccctgaagttccactacacacagaagtctctgtctctgagccccggc BRO2-gaagtgcagctggtgcagtctggcgccgaagtgaagaaacctggcgcctc 39 hIgG4 HCcgtgaaggtgtcctgcaaggcttccggctacacctttaccgactacaacatggactgggtgcgacaggctaccggccagggcctggaatggatcggcgacatcaaccccaactacgagtccaccggctacaaccagaagttcaagggcagagccaccatgaccgtggacaagtccatctccaccgcctacatggaactgtcctccctgcggagcgaggacaccgccgtgtactactgcgccagagaggacgaccacgacgcctttgcttattggggccagggcaccctcgtgaccgtgtcctctgcttctaccaagggcccctccgtgttccctctggccccttgctccagatccacctccgagtctaccgccgctctgggctgcctcgtgaaggactacttccccgagcccgtgacagtgtcttggaactctggcgccctgacctccggcgtgcacacctttccagctgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgactgtgccctccagctctctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccctccttgcccagcccctgaatttctgggcggacccagcgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccaggaagatcccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccttccagcatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgcctccaagccaggaagagatgaccaagaaccaggtgtccctgacctgtctcgtgaaaggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctccttcttcctgtactctcgcctgaccgtggataagtcccggtggcaggaaggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactatacccagaagtccctgtccctgtctctgggaaag BRO2-gaagtgcagctggtgcagtctggcgccgaagtgaaaaaacctggcgcctc 40 hIgG4-cgtgaaggtgtcctgcaaggctagcggctacacctttaccgactacaaca S228P-tggactgggtccgacaggccacaggacagggactcgagtggatcggcgac L445P HCatcaaccccaactacgagagcaccggctacaaccagaagttcaagggcagagccaccatgaccgtggacaagagcatcagcaccgcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagagaggatgatcacgacgcctttgcctattggggccagggcacactggtcaccgttagctctgctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctcaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaatgagtcctagctgg BRO2-gaagtgcagctggtgcagtctggcgccgaagtgaaaaaacctggcgcctc 41 hIgG4-cgtgaaggtgtcctgcaaggctagcggctacacctttaccgactacaaca S228P-NH-tggactgggtccgacaggccacaggacagggactcgagtggatcggcgac L445P HCatcaaccccaactacgagagcaccggctacaaccagaagttcaagggcagagccaccatgaccgtggacaagagcatcagcaccgcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagagaggatgatcacgacgcctttgcctattggggccagggcacactggtcaccgttagctctgctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctcaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgaagttccactacacacagaagagcctctccctgtctccgggtaaa BRO2 LCgacaacgtgctgacccagtcccctgactccctggctgtgtctctgggcga 42gagagccaccatctcttgccgggcctctaagtccgtgcggacctccggctacaactacatgcactggtatcagcagaagcccggccagccccccaagctgctgatctacctggcctccaacctgaagtccggcgtgcccgacagattctccggctctggctctggcaccgactttaccctgaccatcagctccctgcaggccgaggatgccgccacctactactgccagcactccagagagctgccctacacctttggccagggcaccaagctggaaatcaagcggaccgtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtctggcacagcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcga gtgc For SEQ ID NOs:35 and 39, a217g creates N72D mutation

The invention further provides a gene delivery vehicle or vectorcomprising a nucleic acid molecule according to the invention. The genedelivery vehicle or vector can be a plasmid or other bacteriallyreplicated nucleic acid. Such a gene delivery vehicle or vector can beeasily transferred to, for instance, producer cells. The gene deliveryvehicle can also be a viral vector. Preferred viral vectors areadenoviral vectors, lentiviral vectors, adeno-associated viral vectorsand retroviral vectors.

The invention further provides vectors comprising a nucleic acidmolecule or a plurality of nucleic acid molecules in accordance with theinvention. In certain embodiments, a single vector comprises a singlenucleic acid molecule encoding both the VH and the VL domains of themonoclonal antibody or antigen-binding fragment thereof in accordancewith the invention. In certain embodiments, a single vector comprises asingle nucleic acid molecule encoding both the heavy chain (HC) and thelight chain (LC) of the monoclonal antibody or antigen-binding fragmentthereof in accordance with the invention.

In certain embodiments, a first vector comprises a first nucleic acidmolecule encoding the VH domain, and a second vector comprises a secondnucleic acid molecule encoding the VL domain of the monoclonal antibodyor antigen-binding fragment thereof in accordance with the invention. Incertain embodiments, a first vector comprises a nucleic acid moleculeencoding the heavy chain (HC), and a second vector comprises a secondnucleic acid molecule encoding the light chain (LC) of the monoclonalantibody or antigen-binding fragment thereof in accordance with theinvention.

Vectors in accordance with the invention include expression vectorssuitable for use in expressing the monoclonal antibody orantigen-binding fragment thereof by a host cell. Host cells can beeukaryotic or prokaryotic.

The invention provides a host cell comprising a nucleic acid molecule orplurality of nucleic acid molecules encoding an antibody orantigen-binding fragment thereof in accordance with the instantinvention. Alternatively or in addition, the invention provides a hostcell comprising a vector or plurality of vectors encoding an antibody orantigen-binding fragment thereof in accordance with the instantinvention. The nucleic acid molecule or molecules, or similarly thevector or vectors, can be introduced into the host cell using anysuitable technique, including, for example and without limitation,transduction, transformation, transfection, and injection. Various formsof these methods are well known in the art, including, e.g.,electroporation, calcium phosphate transfection, lipofection, cellsqueezing, sonoporation, optical transfection, and gene gun.

In certain embodiments, a host cell is a eukaryotic cell. In certainembodiments, a host cell is a yeast cell. In certain embodiments, a hostcell is an insect cell. In certain embodiments, a host cell is amammalian cell. In certain embodiments, a host cell is a human cell. Incertain embodiments, a host cell is a mammalian cell selected from thegroup consisting of hybridoma cells, Chinese hamster ovary (CHO) cells,NS0 cells, human embryonic kidney (HEK293) cells, and PER.C6™ cells. Theinvention further contemplates other host cells in addition to thosementioned above. Host cells further include cell lines developed forcommercial production of the antibodies and antigen-binding fragmentsthereof in accordance with the invention.

Cell lines provided with the nucleic acid can produce the bindingmolecule/antibody in the laboratory or production plant. Alternatively,the nucleic acid is transferred to a cell in the body of an animal inneed thereof and the binding molecule/antibody is produced in vivo bythe transformed cell. The nucleic acid molecule of the invention istypically provided with regulatory sequences to the express the bindingmolecule in the cell. However, present day homologous recombinationtechniques have become much more efficient. These techniques involve forinstance double stranded break assisted homologous recombination, usingsite-specific double stranded break inducing nucleases such as TALEN.Such or analogous homologous recombination systems can insert thenucleic acid molecule into a region that provides one or more of the incis required regulatory sequences.

The invention further provides an isolated or recombinant cell, or invitro cell culture cell comprising a nucleic acid molecule or vectoraccording to the invention. The invention further provides an isolatedor recombinant cell, or in vitro cell culture cell comprising a bindingmolecule according to the invention. Preferably said cell produces saidbinding molecule. In certain embodiments, said cell secretes saidbinding molecule. In a preferred embodiment said cell is a hybridomacell, a CHO cell, an NS0 cell, a HEK293 cell, or a PER-C6™ cell. In aparticularly preferred embodiment said cell is a CHO cell. Furtherprovided is a cell culture comprising a cell according to the invention.Various institutions and companies have developed cell lines for thelargescale production of antibodies, for instance for clinical use.Non-limiting examples of such cell lines are CHO cells, NS0 cells orPER.C6™ cells. These cells are also used for other purposes such as theproduction of proteins. Cell lines developed for industrial scaleproduction of proteins and antibodies are herein further referred to asindustrial cell lines. The invention provides an industrial cell linecomprising a nucleic acid molecule, a binding molecule and/or antibodyaccording to the invention. The invention also provides a cell linedeveloped for the largescale production of protein and/or antibodycomprising a binding molecule or antibody of the invention. Theinvention also provides the use a cell line developed for the largescaleproduction of a binding molecule and/or antibody of the invention.

Methods of Making Antibodies

The invention further provides a method of making a monoclonal antibodyor antigen-binding fragment thereof in accordance with the invention,comprising culturing a population of host cells according to theinvention under conditions permitting expression of the monoclonalantibody or antigen-binding fragment thereof. In certain embodiments,the method further comprises harvesting said monoclonal antibody orantigen-binding fragment thereof from the culture. Preferably said cellis cultured in a serum-free medium. Preferably said cell is adapted forsuspension growth. Further provided is an antibody obtainable by amethod for producing an antibody according to the invention. Theantibody is preferably purified from the medium of the culture.Preferably said antibody is affinity purified.

Methods of Use

An aspect of the invention is a method of inhibiting activation ofclassical or lectin pathway in a subject, comprising administering to asubject in need thereof an effective amount of the monoclonal antibodyor antigen-binding fragment thereof in accordance with the invention. Incertain embodiments, the subject is a mammal. In certain embodiments,the subject is a mouse, rat, hamster, Guinea pig, rabbit, goat, sheep,pig, cat, dog, horse, or cow. In certain embodiments, a subject is anon-human primate, e.g., a monkey. In certain embodiments, a subject isa human.

The inhibitory effect of the antibody or antigen-binding fragment can beassessed using any suitable method, including, for example, measuringtotal complement activity, a test of hemolytic activity based on theability of a serum sample to lyse sheep erythrocytes coated withanti-sheep antibodies. Decreased hemolysis compared to an untreatedcontrol sample indicates an inhibitory effect of the antibody orantigen-binding fragment. In an embodiment, the untreated control samplecan be a historical sample obtained prior to starting treatment with theantibody or antigen-binding fragment. Generally, a decrease in totalcomplement activity of at least 5% compared to control is indicative ofefficacy. In certain embodiments, a decrease in total complementactivity of at least 10% compared to control is indicative of efficacy.

Diseases that can be treated or prevented by a method or monoclonalantibody or antigen-binding fragment thereof in accordance with theinvention are autoimmune diseases such as experimental allergicneuritis, type II collagen-induced arthritis, myasthenia gravis,hemolytic anemia, glomerulonephritis, idiopathic membranous nephropathy,rheumatoid arthritis, systemic lupus erythematosus, immunecomplex-induced vasculitis, adult respiratory distress syndrome, stroke,xenotransplantation, allotransplantation, multiple sclerosis, burninjuries, extracorporeal dialysis and blood oxygenation, inflammatorydisorders, including sepsis and septic shock, toxicity induced by the invivo administration of cytokines or mAbs, antibody-mediated rejection ofallografts such as kidney allografts, multiple trauma,ischemia-reperfusion injuries, and myocardial infarction.

Individuals suffering from a disease involving complement-mediateddamage or at risk of developing such complement-mediated damage can betreated by administering an effective amount of a monoclonal antibody orantigen-binding fragment thereof in accordance with the invention to anindividual in need thereof. Thereby the biologically activecomplement-derived peptides are reduced in the individual and the lyticand other damaging effects of complement on cells and tissues isattenuated or prevented. By “effective amount” is meant an amountsufficient to achieve a desired biological response. In an embodiment,by “effective amount” is meant an amount of a monoclonal antibody orantigen-binding fragment thereof in accordance with the invention thatis capable of inhibiting complement activation in the individual.

Treatment (prophylactic or therapeutic) will generally consist ofadministering the monoclonal antibody or antigen-binding fragmentthereof in accordance with the invention parenterally together with apharmaceutical carrier, for example intravenously, subcutaneously, orlocally. The administering typically can be accomplished by injection orinfusion. The dose and administration regimen of the monoclonal antibodyor antigen-binding fragment thereof in accordance with invention willdepend on the extent of inhibition of complement activation aimed at.Typically, for monoclonal antibodies of the invention, the amount willbe in the range of 2 to 20 mg per kg of body weight. For parenteraladministration, the monoclonal antibody or antigen-binding fragmentthereof in accordance with the invention will be formulated in aninjectable form combined with a pharmaceutically acceptable parenteralvehicle. Such vehicles are well-known in the art and examples includesaline, dextrose solution, Ringer's solution and solutions containingsmall amounts of human serum albumin.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, micro-emulsion, liposome, or other orderedstructure suitable to high drug concentration. Examples of suitableaqueous and non-aqueous carriers which may be employed in thepharmaceutical compositions of the invention include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

The pharmaceutical compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of presence of microorganisms may be ensured both bysterilization procedures and by the inclusion of various antibacterialand antifungal agents, for example, paraben, chlorobutanol, phenol,sorbic acid, and the like. It may also be desirable to includeisotonicity agents, such as sugars, polyalcohols such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.Pharmaceutically-acceptable antioxidants may also be included, forexample (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Sterile injectable solutions can be prepared by incorporating the mAb orfragments thereof in the required amount in an appropriate solvent withone or a combination of ingredients e.g. as enumerated above, asrequired, followed by sterilization microfiltration. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients e.g. from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Prolonged absorption of the injectable anti-C2 mAbs or fragments thereofcan be brought about by including in the composition an agent thatdelays absorption, for example, monostearate salts and gelatin.

The mAbs of fragments thereof can be prepared with carriers that willprotect the compound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for the preparation of such formulations are generally known tothose skilled in the art. See, e.g., Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978.

The pharmaceutical compositions can be administered with medical devicesknown in the art.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time, orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation.

Actual dosage levels of the mAbs or fragments thereof in thepharmaceutical compositions of the present invention may be varied so asto obtain an amount of the active ingredient which is effective toachieve the desired therapeutic response for a particular patientwithout being toxic to the patient.

In one embodiment, the monoclonal antibodies according to the inventioncan be administered by infusion in a weekly dosage of from 10 to 500mg/m², such as of from 200 to 400 mg/m². Such administration can berepeated, e.g., 1 to 8 times, such as 3 to 5 times. The administrationmay be performed by continuous infusion over a period of from 2 to 24hours, such as of from 2 to 12 hours.

In yet another embodiment, the mAbs or fragments thereof or any otherbinding molecules disclosed in this invention, can be administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

The present invention will now be illustrated with reference to thefollowing examples, which set forth particularly advantageousembodiments. However, it should be noted that these embodiments aremerely illustrative and are not to be construed as restricting theinvention in any way.

EXAMPLES Example 1: Removal of a Glycosylation Site from an Anti-C2bMonoclonal Antibody

BRO2-glyc-IgG4

U.S. Pat. No. 9,944,717 discloses a murine inhibitory anti-C2bmonoclonal antibody (mAb). From this lead, four humanized variants,comprising different heavy chain variable domains (VH1, VH2, VH3, orVH4) and kappa light chain variable domains (VK1, VK2, VK3, or VK4),were generated using the Composite Human Antibody technology of AntitopeLtd (Cambridge, UK). Based on in silico analysis, the risk ofimmunogenicity for each of the humanized VH and VK sequences waspredicted. As shown in Table 7, the lowest risk for immunogenicity,along with the highest percentage of identity to the closest humangermline variant, was predicted when VH4 was paired with VK3 or VK4.This observation was based on the lowest number of promiscuous bindingpeptides to human MHC class II. VH4 was preferred because of its higherpercentage of identity against the closest human germline. In addition,based on binding and potency, VH4/VK3 was selected as the anti-human C2bhumanized lead antibody and is referred to herein as BRO2-glyc-IgG4.

TABLE 7 Risk for immunogenicity ranked 1 (=lowest) to 5 (=highest) (highaffinity priority over moderate affinity) and sequence identity to theclosest human germline Identity Identity High Moderate to IGHV1- HighModerate to IGKV4- VH Affinity Affinity Ranking 8*01 VL AffinityAffinity Ranking 1*01 WT 1 2 5 79.3% WT 6 5 5 80.0% VH1 0 3 4 86.2% VK13 3 3 92.5% VH2 0 2 1 90.8% VK2 3 3 3 95.0% VH3 0 2 1 93.1% VK3 3 2 196.3% VH4 0 2 1 95.4% VK4 3 2 1 97.5%

SDS-PAGE analysis of variants of BRO2-glyc-IgG4 revealed a double bandand band shift in the VH3 and VH4 variants. This shift was hypothesizedto arise from a potential glycosylation site (motif NXS) at residues72-74 (Kabat numbering) in framework region 3 (FR3) of VH3 and VH4.Because this potential glycosylation site could result in heterogeneitynot only of antibody product expressed from mammalian cell lines, butalso of antibody function, the potential glycosylation site was removed.The glycosylation site was removed by site-directed mutagenesis togenerate an N72D variant of the VH, referred to herein as either VH3.2or VH4.2. The N72D mutation removed the altered band pattern observed inVH3 and VH4 (FIG. 1), confirming that the double band and band shift wascaused by glycosylation and heterogeneity in the heavy chain.

To further determine whether variant VH4.2, which is the same VH asBRO2-glyc-IgG4 but without the glycosylation site in FR3, demonstratedimproved characteristics compared to the heterogeneously glycosylatedparent mAb BRO2-glyc-IgG4, thermotolerance of each antibody wasdetermined.

To test thermotolerance, humanized variants were treated with anincreasing temperature from 55° C. up to 75° C. with Thermocycler(Biometra). Residual binding capacity was analyzed on Biacore 3000 on aCMS Chip directly coated with human C2 purified from serum (3500 RU,Complement Technologies Cat # A112, lot #20). Data were analyzed usingthe BIAevaluation software. The slope of specific binding of eachvariant was determined with the BIAevaluation software, general fit fromthe linear phase of the sensorgram (started at 5 seconds after the startof injection and stopped after 11 seconds). Then percentage of activitywas calculated, using the mean of the slope obtained for the 59° C.,56.9° C., 55° C. and 4° C. temperatures as 100% activity. Finally, thepercentage of activity was plotted in GraphPad Prism (Log (agonist) vsresponse, variable slope (4 parameters)). The temperature where theantibody lost 50% of its binding capacity (TM50) is shown in Table 8below.

BRO2-IgG4

Both variants without the glycosylation site present in BRO2-glyc-IgG4demonstrated improved thermotolerance (Table 8). BRO2-glyc-IgG4exhibited a TM50 of 64.0° C. VH4.2/VK3 (also referred to herein asBRO2-IgG4) exhibited a TM50 of 65.0 or 65.1° C. in two independentexperiments. VH4.2/VK4 exhibited a TM50 of 65.2 or 65.4° C. in twoindependent experiments.

TABLE 8 Percent Identity to closest human germline sequences andthermotolerance of Anti-C2b Monoclonal Antibodies BRO2-glyc- VH4.2/VK3IgG4 (BRO2-IgG4) VH4.2/VK4 % Identity to closest 95.8 95.3 95.9 humangermline sequences % Homology to closest 97.0 97.0 97.6 human germlinesequences Thermotolerance 64.0 65.0; 65.1 65.2; 65.4 (TM50, ° C.)

Example 2: Preparation of Non-glycosylated IgG4 and Non-glycosylatedIgG1 Variants

BRO2-IgG4-NH

Antibodies with pH-dependent antigen binding dissociate bound antigen inacidic endosomes after internalization into cells. Consequently,released antigen is trafficked to the lysosome and degraded, whereas thedissociated antibody, free of antigen, is recycled back to plasma byFcRn. The recycled free antibody can bind to another target antigen. Byrepeating this cycle, a pH-dependent antigen-binding antibody can bindto the target antigen more than once and therefore improve theneutralizing capacity of the antibody. This process can further beimproved when an antibody is equipped with NHance® (NH) technology(argenx, Belgium) that enhances the binding of the antibody to FcRn atacidic endosomal pH (pH 6.0) but not at neutral pH (pH 7.4). Therefore,amino acids in the Fc region of BRO2-IgG4 were mutated to alterpH-dependent binding to FcRn (H433K, N434F). The resulting antibody isreferred to herein as BRO2-IgG4-NH.

BRO2-IgG1-NH and

BRO2-IgG1-LALA-NH (ARGX-117)

The effect of immunoglobulin subclass on efficacy was also examined. Afurther NHance® variant was prepared in a human IgG1 background(BRO2-IgG1-NH). Antibody effector functions can be further diminished bymutations in the Fc region that alter binding of the antibody to Fcγreceptors. Therefore, amino acid substitutions L234A and L235A (“LALA”)were incorporated into BRO2-IgG1-NH to yield BRO2-IgG1-LALA-NH, alsoreferred to herein as ARGX-117.

His1-IgG1-LALA-NH

To determine if pH dependency of BRO2-IgG1-LALA-NH could be improved toextend its pharmacokinetic and pharmacodynamic (PK/PD) effects in vivo,an amino acid in the VK of the BRO2-IgG1-LALA-NH antibody was mutated tohistidine (G29H, mutant VK referred to herein as Vk3m3). The resultingantibody is referred to herein as His1-IgG1-LALA-NH.

His1-IgG4

Similarly, to determine if pH dependency of BRO2-IgG4 could be improvedto extend its PK/PD effects in vivo, an amino acid in the VK of theBRO2-IgG4 antibody was mutated to histidine (G29H, mutant VK referred toherein as Vk3m3). The resulting antibody (VH4.2/Vk3m3) is referred toherein as His1-IgG4.

His1-IgG4-NH

To examine the effect of recycling on antibody efficacy, the NHance®mutations were incorporated into the His1-IgG4 (VH4.2/Vk3m3) antibody.The resulting antibody is referred to herein as His1-IgG4-NH.

His2-IgG4-NH

To determine if pH dependency of BRO2-IgG4-NH could be improved toextend its PK/PD effects in vivo, an amino acid of the VH4 of theBRO2-IgG4-NH antibody was mutated to histidine (K26H, VH mutant referredto herein as VH4.2m12). Additionally, the VK3 light chain of theBRO2-IgG4-NH antibody was replaced with the VK4 light chain mentionedabove, and a second amino acid was mutated to histidine (G29H, VK4mutant referred to herein as VK4m3). The resulting antibody(VH4.2m12/VK4m3) is referred to herein as His2-IgG4-NH.

Example 3: Efficacy Improvements in Non-glycosylated BRO2 Variants

Total Pharmacokinetics (PK)

Cynomolgus monkeys (n=2, 1 male and 1 female per group) were randomlyassigned into separate treatment groups in accordance with Table 9below.

TABLE 9 Treatment Group Assignments Animal Group Antibody No. 1 BRO2glyc-IgG4 1 2 2 Negative Control 3 4 3 BRO2-IgG4 5 6 4 BRO2-IgG4-NH 7 85 BRO2-IgG1-LALA-NH 9 10 6 His1-IgG4 11 12 7 His1-IgG4-NH 13 14 8His1-IgG1-LALA-NH 15 16 9 His2-IgG4-NH 17 18

A serum sample was obtained from each monkey one day prior to receivingtest antibody (day −1, or “PRE”). Then on day 1 (d1), each monkeyreceived a single intravenous injection of 5 mg/kg test antibody inaccordance with Table 9. Serum samples were then obtained from eachmonkey serially over up to 60 days (to d60).

For PK of total antibody (total PK), a microtiter plate was coatedovernight at 4° C. with 100 μL goat anti-human IgG (Bethyl; A80-319A) at5 μg/mL. Plates were washed 3 times with at least 200 μL PBS-0.05%Tween20 and subsequently blocked with 200 μL PBS-2% BSA for 2 hours atroom temperature (RT). After washing the plates 3 times with at least200 μL PBS-0.05% Tween20, serum samples, standard and QC samples(prepared in pooled naïve cynomolgus monkey serum) were applied induplicate at 100-fold dilution or more and diluted in 100 μL PBS-0.2%BSA-1% pooled naïve cynomolgus monkey serum. For each antibody, its ownfrozen standards and QC samples were applied in duplicate (the samebatch of antibody was used as the batch that was injected in themonkeys). The negative control antibody is an antibody that binds anon-C2 complement component. Incubation was done at RT for 2 hourswhilst shaking the plate. After washing the plates 5 times with at least200 μL PBS-0.05% Tween20, 100 μL horseradish peroxidase (HRP)-labeledmouse anti-human IgG kappa (Southern Biotech, 9230-05) was diluted260,000-fold in PBS 0.2% BSA and applied to the wells for 1 hour at RT.The plates were washed 5 times with at least 200 μL PBS-0.05% Tween20and staining was done with 100 μL 3,3′,5,5′-tetramethylbenzidine (TMB)and stopped after 10 minutes with 100 μL 0.5 M H₂SO₄ (CHEM LAB, Cat #CL05-2615-1000). The OD was measured at 450 nm and GraphPad Prism wasused to back calculate the concentration of samples (each using its ownstandard).

Results are shown in Table 10 and a comparison of glycosylatedBRO2-glyc-IgG4 with non-glycosylated BRO2-IgG4 is shown in FIG. 2. Inthe total PK assay, concentrations of non-glycosylated BRO2-IgG4 weregenerally greater than those of glycosylated BRO2-glyc-IgG4. Thisimprovement in total PK was completely unexpected and represents animportant further advantage of the non-glycosylated antibody.

TABLE 10 Total PK Total PK (μg/mL) BRO2-glyc-IgG4 BRO2-IgG4 average Stdaverage Std Monkey 1 Monkey 2 M1&M2 Dev Monkey 5 Monkey 6 M5&M6 Dev 15min 107.1 99.9 103.4 5.3 167.0 166.7 162.5 5.6 1 h 103.9 99.9 102.2 1.7172.9 158.4 168.1 13.3 2 h 95.4 89.0 94.8 2.8 151.1 150.9 147.6 3.8 4 h91.4 92.2 90.0 2.5 134.6 132.3 132.7 1.2 6 h 86.5 86.0 85.4 1.6 134.3128.9 133.6 5.5 24 h 56.1 55.5 58.3 1.2 99.6 97.6 85.9 1.9 Day 2 47.947.2 47.3 0.6 77.6 78.3 68.7 9.3 Day 4 34.8 39.8 34.1 2.7 58.4 55.8 60.84.8 Day 7 26.6 25.1 26.0 0.6 41.5 33.6 34.9 7.6 Day 11 16.3 14.1 16.21.3 28.1 20.9 24.5 5.1 Day 15 9.8 8.1 9.3 0.9 18.1 11.2 13.0 5.1 Day 196.7 5.1 6.5 1.1 12.8 6.5 8.3 2.5 Day 23 4.4 3.2 4.4 0.8 9.8 4.3 7.1 3.9Day 27 3.2 1.9 3.1 0.7 6.5 2.2 3.9 3.0 Day 31 2.2 1.5 2.3 0.4 4.7 1.02.9 2.6

Free C2

Cynomolgus monkeys (n=2, 1 male and 1 female per group) received asingle intravenous injection of 5 mg/kg test antibody, as describedabove.

In this assay a microtiter plate was coated overnight at 4° C. with 100μL 2.5 μg/mL mouse anti-human C2 monoclonal antibody mAb32 (anti-C2#32m-IgG @ 3.31 mg/mL, 0.2 μm PBS, LC-12/05-166, 12-apr-13). This antibodybinds to a different epitope on C2 than BRO2. Plates were washed 3 timeswith at least 2004 PBS-0.05% Tween20 and subsequently blocked with 200μL PBS-2% BSA (pH 7.4) for 2 hours at RT. In the meantime, samples,frozen standard (specific for each antibody, prepared in pooled naïvecynomolgus monkey serum) and frozen QC samples (prepared in pooled naïvecynomolgus monkey serum) were thawed and diluted 6.7-fold in 80 μLPBS-0.2% BSA. 40 μL biotinylated anti-C2 VH4/VK3 was added at 0.6 μg/mL.Each sample was made in duplicate. 100 μL of the mixture was transferredimmediately to the washed coated plate after addition of thebiotinylated antibody. The plate was incubated for 2 hours at RT, washed5 times with at least 2004 PBS-0.02% Tween20, and 100 μL strep-HRP(Jackson, 016-030-084) was added at 300,000-fold dilution in PBS-0.2%BSA. After 1 hour incubation at RT, the plates were washed 5 times withat least 200 μL PBS-0.05% Tween20 and staining was done with 100 μL TMB(Calbiochem, CL07) and stopped after 10 minutes with 100 μL 0.5 M H₂SO₄(CHEM LAB, Cat # CL05-2615-1000). The OD was measured at 450 nm and usedto determine C2 levels.

Sera from the following monkeys were first tested together using thefree C2 assay performed on different days: monkeys 1 and 2; monkeys 3and 4; monkeys 5 and 6; monkeys 7, 8, 9, and 10; monkeys 11, 12, 15, and16; and monkeys 13, 14, 17, and 18.

The levels of free C2 for all monkeys are shown in FIGS. 3A-3I and inTable 11.

As expected, for monkeys 3 and 4 there was no decline in free C2, asthese monkeys were dosed with a negative control antibody. For allmonkeys treated with the BRO2 variants, free C2 levels were very lowuntil after day 2.

For the monkeys receiving BRO2-glyc-IgG4 (monkeys 1 and 2) and BRO2-IgG4(monkeys 5 and 6), C2 levels went back up beginning at day 4 and wereback to baseline levels by day 31. Monkeys 5 and 6, treated withnon-glycosylated antibody, consistently displayed lower free C2 levelsthan those treated with BRO2-glyc-IgG4 (FIG. 3C, Table 11).

For all other monkeys, excluding those with anti-drug antibodies (ADA,marked by a * in FIGS. 3D-3I), C2 levels increased much more slowly, andC2 levels did not return to baseline even by day 31.

FIG. 4 shows a blow up (log scale) of the free C2 levels (OD 450 nm) forthe average of the 2 monkeys of each group. Free C2 levels were lowerfor BRO2 variants than for His1 variants.

Monkey 10, injected with BRO2-IgG1-LALA-NH (ARGX-117), had the lowestlevels of C2 at all time points tested. Comparison of free C2 levelsfrom monkeys 5 and 6, 9 and 10, and 15 and 16 out to 60 days can be seenin FIG. 5. Monkey 10 also had the best total PK (see above). The rawdata is shown in Table 11, and average data comparing the glycosylatedand non-glycosylated variants is shown in Table 12.

TABLE 11 Free C2 (OD450 nm) for all antibodies Time Point M 1 M 2 M 3 M4 M 5 M 6 M 7 M 8 M 9 M 10 M 11 M 12 M 13 M 14 M 15 M 16 M 17 M 18 Day−5 0.773 0.770 0.797 0.930 0.696 0.705 0.439 0.532 0.602 0.626 0.6560.643 0.824 0.805 0.756 0.755 0.789 0.935 Day 0 0.707 0.716 0.813 0.9070.740 0.657 0.507 0.531 0.578 0.604 0.620 0.687 0.847 0.792 0.772 0.7500.834 1.043 15 m 0.031 0.028 0.879 0.979 0.016 0.018 0.016 0.014 0.0150.013 0.029 0.033 0.032 0.038 0.033 0.028 0.050 0.055 1 h 0.033 0.0280.914 1.055 0.017 0.021 0.014 0.013 0.014 0.013 0.030 0.033 0.035 0.0370.034 0.031 0.050 0.062 2 h 0.034 0.030 0.874 0.997 0.018 0.020 0.0160.014 0.015 0.015 0.031 0.031 0.034 0.043 0.034 0.030 0.052 0.063 4 h0.033 0.027 0.887 1.000 0.017 0.020 0.017 0.015 0.016 0.016 0.037 0.0340.036 0.044 0.037 0.032 0.054 0.062 6 h 0.034 0.030 0.958 1.035 0.0150.019 0.015 0.014 0.015 0.017 0.032 0.034 0.035 0.043 0.034 0.034 0.0520.071 Day 1 0.046 0.045 0.917 0.923 0.021 0.025 0.019 0.018 0.020 0.0200.041 0.038 0.046 0.054 0.048 0.044 0.064 0.084 Day 2 0.060 0.061 0.8720.920 0.027 0.030 0.021 0.018 0.023 0.022 0.075 0.040 0.050 0.052 0.0530.043 0.068 0.090 Day 4 0.125 0.102 0.833 0.886 0.037 0.048 0.026 0.0210.035 0.024 0.088 0.056 0.063 0.064 0.056 0.055 0.080 0.096 Day 7 0.1740.169 0.853 0.899 0.072 0.110 0.033 0.025 0.050 0.028 0.119 0.085 0.0750.075 0.070 0.071 0.080 0.099 Day 11 0.257 0.265 0.862 0.847 0.127 0.1930.050 0.033 0.092 0.033 0.172 0.105 0.088 0.088 0.090 0.087 0.102 0.134Day 15 0.364 0.375 0.840 0.834 0.177 0.290 0.065 0.043 0.138 0.043 0.3150.138 0.086 0.096 0.094 0.109 0.133 0.147 Day 19 0.418 0.471 0.807 0.8640.256 0.406 0.083 0.031 0.194 0.051 0.289 0.157 0.157 0.106 0.113 0.1270.143 0.167 Day 23 0.517 0.562 0.820 0.897 0.327 0.469 0.100 0.059 0.2550.062 0.351 0.225 0.339 0.133 0.126 0.148 0.176 0.192 Day 27 0.597 0.6330.818 0.921 0.378 0.537 0.124 0.146 0.292 0.071 0.418 0.230 0.492 0.2550.140 0.170 0.199 0.231 Day 31 0.633 0.663 0.841 0.934 0.431 0.599 0.1530.125 0.364 0.098 0.511 0.280 0.605 0.522 0.163 0.205 0.238 0.244

TABLE 12 Average Free C2 of Glycosylated and Non-Glycosylated AntibodiesFree C2 (OD 450 nm) BRO2-glyc-IgG4 BRO2-IgG4 average Standard averageStandard Monkey 1 Monkey 2 M1&M2 Deviation Monkey 5 Monkey 6 M5&M6Deviation Day −5 0.773 0.77 0.772 0.002 0.696 0.705 0.701 0.006 Day 00.707 0.716 0.712 0.006 0.74 0.657 0.699 0.059 15 min 0.031 0.028 0.0300.002 0.016 0.018 0.017 0.001 1 h 0.033 0.028 0.031 0.004 0.017 0.0210.019 0.003 2 h 0.034 0.03 0.032 0.003 0.018 0.02 0.019 0.001 4 h 0.0330.027 0.030 0.004 0.017 0.02 0.019 0.002 6 h 0.034 0.03 0.032 0.0030.015 0.019 0.017 0.003 Day 1 0.046 0.045 0.046 0.001 0.021 0.025 0.0230.003 Day 2 0.06 0.061 0.061 0.001 0.027 0.03 0.029 0.002 Day 4 0.1250.102 0.114 0.016 0.037 0.048 0.043 0.008 Day 7 0.174 0.169 0.172 0.0040.072 0.11 0.091 0.027 Day 11 0.257 0.265 0.261 0.006 0.127 0.193 0.1600.047 Day 15 0.364 0.375 0.370 0.008 0.177 0.29 0.234 0.080 Day 19 0.4180.471 0.445 0.037 0.256 0.406 0.331 0.106 Day 23 0.517 0.562 0.540 0.0320.327 0.469 0.398 0.100

As these assays for the different monkeys just described were run ondifferent days, the analysis was repeated for a select number of timepoints (pre, 4 hours, days 1, 2, 4, 11, and 27) where sera from allmonkeys were put on a single plate (FIGS. 6A-6D). The pre-samples werealso tested with and without addition of excess BRO2 (500 μg/mL).

The ODs of the pre-samples were comparable for all monkeys, indicatingthat free C2 levels in the different monkeys were comparable (FIG. 6A).When the pre-samples were pre-incubated with 500 μg/mL BRO2, all signalsdropped to an OD of 0.013-0.015 (FIGS. 6A and 6B). Such low OD valueswere not obtained for any of the PK samples, indicating that at no timepoint was free C2 completely depleted. The lowest levels were obtainedat 4 hours, and they were the lowest (OD between 0.02 and 0.03) for theBRO2 variants (monkeys 5, 6, 7, 8, 9, and 10, FIG. 6C). Interpretationof the results at day 11 and day 27 was hampered by ADA (anti-drugantibodies) that was observed in several of the monkeys (FIG. 6D).

Immunogenicity

Cynomolgus monkeys (n=2, 1 male and 1 female per group) received asingle intravenous injection of 5 mg/kg test antibody, as describedabove. Serum samples obtained from all monkeys were tested for ADA(anti-drug antibodies) from baseline (pre-exposure) until day 31 (FIGS.7A-7P), and serum samples obtained from monkeys 5 and 6, 9 and 10, and15 and 16 were further tested until day 59 (FIGS. 8A-8F).

Immunogenicity was determined by coating a microtiter plate with 100 μLof 1 μg/mL of the respective antibody overnight at 4° C. Plates werewashed 3 times with at least 200 μL PBS-0.05% Tween20 and subsequentlyblocked with 200 μL PBS-1% casein for 2 hours at RT. After washing theplates 3 times with at least 200 μL PBS-0.05% Tween20, serum sampleswere diluted 20-fold or more in 100 μL PBS-0.1% casein and incubated inthe coated wells for 2 hours at room temperature (RT). After washing theplates 5 times with at least 200 μL PBS-0.05% Tween20, 100 μLanti-monkey IgG-HRP (Southern Biotech #4700-05) was added to the wellsat a 8000-fold dilution for 1 hour at RT. The plates were washed 5 timeswith at least 200 μL PBS-0.05% Tween20 and staining was done with 100 μLTMB and stopped after 10 minutes with 100 μL 0.5 M H₂SO₄ (CHEM LAB, Cat# CL05-2615-1000). The OD was measured at 450 nm. Representative resultsare shown in FIGS. 7A-7P.

A clear ADA response was observed for monkeys 8 (BRO2-IgG4-NH), 11(His1-IgG4), 13 and 14 (His1-IgG4-NH), and 16 (His1-IgG1-LALA-NH) (FIGS.7F, 7I, 7K and 7L, and 7N, respectively). Indeed, the signal obtained inELISA after injection of the antibody as compared to the baseline(“PRE”) signal (before injection of the antibody) was increased at least2-fold.

For monkeys 11, 13, and 16 (FIGS. 7I, 7K, and 7N, respectively), ADA wasobserved as of day 11; for monkey 8 (FIG. 7F), as of day 15; and formonkey 14 (FIG. 7L), as of day 19.

For monkey 9 (BRO2-IgG1-LALA-NH) (FIG. 7G), an increase in signal wasobserved for all samples post injection of the antibody, but the signalin the baseline sample was already high and the increase over time waslow (about 1.5-fold).

For monkey 5 (FIG. 7C) an unusually high signal was observed in thebaseline sample before injection of the antibody. This signal was alsohigher than the signals of the later timepoints. This may be explainedby the interference of the antibody (present in the serum) with theassay. It was therefore not possible to determine if there was an ADAresponse in this monkey.

Example 4: Isoelectric Point (pI)

Igawa et al. (Protein Eng Des Sel 2010, 23(5):385-392), studying VHmutants of certain IgG1 monoclonal antibodies, reported a strongpositive correlation between isoelectric point (pI) and monoclonalantibody clearance and a negative correlation between pI and monoclonalantibody half-life. In this example, the pI of various forms ofanti-human C2b were determined. Results are shown in Table 13.

TABLE 13 Apparent pI of Anti-human C2b Monoclonal Antibodies VH4.2-VH4.2- VH4.2-IgG1- IgG4- IgG4-HN- LALA-HN-IAP2VK3 Peak IAP2VK3 IAP2VK3(ARGX-117) Calculated Acidic 3 7.02 7.14 Apparent pI Acidic 2 7.10 7.24n = 3 Acidic 1 7.16 7.32 8.29 Main Peak 7.20 7.35 8.43 Basic 1 7.30 7.458.57 Basic 2 7.42 7.58All three antibodies tested are without glycosylation in VH. As shown inTable 13, the pI of ARGX-117 was found to be significantly greater thanthe pI of closely related IgG4 antibodies. The observed pI of ARGX-117is expected to be manifested as enhanced potential for so-called antigensweeping.

Example 5: Domain Mapping by Western Blotting and Surface PlasmonResonance (SPR) Analysis

Binding characteristics of ARGX-117 were assessed by Western blottingand by Surface Plasmon Resonance (SPR) analysis, as depicted in FIG. 1.Western blotting results revealed that ARGX-117 binds to C2 and C2b, asdepicted in FIG. 9A. The binding characteristics of ARGX-117 werefurther studied by SPR, using the Biacore 300, by coating C2 (SEQ ID NO:21) on the solid phase with different concentrations of Fabs of ARGX-117used as eluate, as depicted in FIG. 9B. Affinities were calculatedassuming 1:1 binding between the Fab and C2 and yielded a Kd of about0.3 nM. In order to study the mechanism of action by ARGX-117, SPRanalysis was performed, mimicking the formation of C3 convertase(C4bC2a) with biotinylated C4 immobilized to streptavidin-coated chips,as depicted in FIG. 9C. When C2 was added in flowing buffer, alone orpreincubated with the control mAb, C2 binding was observed on the chip.Pre-incubation with anti-C2 clone 63 (i.e., anti-C2-63) resulted inhigher signal, presumably because this mAb form complexed to C2 andC2:mAb complexes bind together resulting in higher molecular mass andhigher SPR signal. When C2 was pre-incubated with ARGX-117, binding ofC2 to C4b was greatly reduced. The initial interaction of C2 to C4b isthought to be initiated by the C2b domain (SEQ ID NO: 44). Thereafterthe large C2a domain (SEQ ID NO: 43) takes over and this interaction iscrucial in the formation of the C3 convertase complex. The results fromthis experiment suggest that ARGX-117 inhibits C2 by inhibiting bindingto C4b.

To further understand the mechanism of action of C2 inhibition byARGX-117, C2 was first allowed to bind to C4b immobilized onstreptavidin chips, and after stabilization by flowing buffer only,samples were flown, as depicted in FIG. 9D. Running buffer or controlhuman IgG4 mAb targeting an irrelevant soluble antigen (i.e.,anti-Factor XI (anti-FXI)) resulted in some signal decrease, whichnormalized after injection ceased. Injection of anti-C2-63 resulted inincreased signal, suggesting that this mAb is able to bind to C3convertase (C4bC2a). This is in line with the predicted binding model ofC2 to C4b, which suggests that after binding on C2, the C2a domain isstill largely available. Interestingly, ARGX-117 demonstrated a strongbinding to C3 convertase that was followed by a rapid dissociation.These results suggest that ARGX-117 is able to bind C2, but that thisbinding is very unstable, likely affecting C2 in a way that facilitatesactivation. These results also suggest that ARGX-117 would be releasedtogether with C2b from the C2 molecule.

Example 6: Domain Mapping Using Domain Swap Mutants of C2 and Factor B

In order to map the epitope of anti-C2-5F2.4, advantage was taken of thefact that anti-C2-5F2.4 does not cross-react with Factor B (FB; SEQ IDNO: 50) and that C2 and FB are highly homologous proteins that havesimilar domain structure. Both proteins comprise a small fragment, and alarge fragment. The small fragment in complement C2 is called C2b (SEQID NO:44), and the small fragment in Factor B is called FBa (SEQ ID NO:51). The small fragment in each comprises three Sushi domains (CCPdomains). The large fragment in each comprisesa von Willebrand Factortype A domain (VWFA) and a Peptidase 51 domain on, as shown in FIG. 10.Domain swap mutants included a C-terminal FLAG tag.

To generate the various swap mutants, DNA constructs for C2, FB, and theten domain swap mutants were obtained from GenScript. DNA was heatshock-transformed into competent E. coli cells (ThermoFisher). Cellswere streaked on agar plates and grown for 16 hours at 37° C. Thirteenbottles of 200 mL LB (Luria Broth) medium were prepared (MP Bio) andautoclaved. 300 μL ampicillin (100 mg/mL) was added to each bottle.Pre-cultures were started with 3 mL LB medium for each construct. After6 hours, the pre-cultures were transferred into the bottles and grownfor 16 hours at 37° C. with agitation. DNA was purified from bacterialpellets by a plasmid DNA purification kit according the manufacturer'sinstructions (MaxiPrep, NucleoBond PC 500, Macherey-Nagel) andreconstituted in TE buffer. Plasmid DNA concentration was determined byNanoDrop and was set to 1 μg/μL. The integrity of the plasmids wasverified by restriction analysis. For each construct 1 μL plasmid DNAand 9 μL restriction enzyme-mix (Pstl and Pvull) were mixed andincubated for 2 hours at 37° C. The DNA was analyzed on a 1% agarose gelafter 1 hour running at 100 V using Bio-Rad ChemiDoc MP system. DNAconstructs for the fine mapping (see below) were handled the same waybut their integrity was checked by sequencing.

The mutant proteins were generated by transient transfection in HEK293Tcells. HEK cells were cultured in complete DMEM (DMEM (Gibco)supplemented with 10% fetal calf serum (FCS) and 1%penicillin/streptomycin (P/S)). One day prior to transfection, cells oftwo flasks were seeded into fifteen 10 cm² culture dishes (GreinerBio-One). Before the transfection, 21 mL of empty DMEM medium was mixedwith 630 μL polyethylenimine (P-Pei, Polysciences, Inc.). As controls anempty plasmid PF45 pcDNA3.1 and PF146 H2B GFP were transfected. 15 μgplasmid DNA was incubated in 1500 μL empty medium-P-Pei mix for 20minutes in Eppendorf-tubes. The transfection mix was carefully added tothe cells and the medium was mixed by pipetting up and down. After 8hours the medium was changed to 15 mL empty medium. After 3 days thecells were checked for GFP expression with a fluorescence microscope.Supernatants were collected on day 4 and were filter-sterilized by a0.22 μm filter (Sartorius) and concentrated with a Vivaspin column(Sartorius) to approximately one-third of the original volume. Domainswap mutants were concentrated with 30,000 MWCO columns, and C2b mutantsfor fine mapping were concentrated with 10,000 MWCO columns. Allsupernatants were stored at −20° C. and were analyzed also by SDS-PAGEand anti-FLAG Western Blot.

To verify expression of the various constructs, an anti-FLAG-tag ELISAassay was carried out. Microplates (Maxisorp, NUNC, Cat #439454) werecoated overnight with 100 μL of HEK293T supernatants 5× diluted in PBSor undiluted (for domain swap mutants and fine mapping mutants,respectively). After washing 4 times with PBS and 0.05% Tween-20, 100μL/well of 1 μg/mL anti-FLAG Ab (clone M2, Sigma-Aldrich) in PBS and0.1% Tween-20 (PBST) was added and incubated for 1 hour at roomtemperature (RT) with agitation. As detection Ab, 100 μL/well ofhorseradish peroxidase (HRP)-labeled goat anti-mouse-IgG (Santa CruzBiotechnology, Cat # sc-2005, 1000× dil.) was added in PBST andincubated for 1 hour at RT. After a final washing step, 100 μL/well TMB(Invitrogen, Cat # SB02) was added as substrate, the reaction wasstopped after a few minutes with 100 μL/well HCl (Fischer, Cat #J/4320/15) and the absorbance was read at 450 nm (BioRad, iMarkMicroplate reader).

Anti-FLAG ELISA detected proteins in the supernatant for all mutants,except for C2-(FB-Pep1), as depicted in FIG. 11. The variation betweenthe mutants can be explained by the different production or by thedifferent detection efficacy by anti-FLAG mAb after coating.

Next, the recognition of the swap mutants by the anti-C2-5F2.4 antibodywas investigated. To this effect, microplates (Maxisorp, NUNC, Cat#439454) were coated overnight with 2 μg/mL anti-C2-5F2.4 in 100 μL PBS.Plates were washed 4 times with PBS with 0.05% Tween-20 and blocked with200 μL PBS with 0.1% Tween-20 with 1% bovine serum albumin (BSA)(PBST-BSA) for 1 hour at RT. After washing, 100 μL culture supernatantcontaining mutants were added 20× diluted in PBST-BSA and incubated for2 hours at RT with agitation. After washing, as detection antibody 1μg/mL biotinylated anti-FLAG (clone M2, Sigma-Aldrich) was added inPBST-BSA for 1 hour at RT. The plate was washed and 1 μg/mLstreptavidin-POD conjugate (Roche, Cat #11089153001) was added andincubated in the dark for 30 minutes. The plate was washed and 100μL/well TMB (Invitrogen, Cat # SB02) was added as substrate, andreaction was stopped after a few minutes with 100 μL/well HCl (Fischer,Cat # J/4320/15). Absorbance was measured at 450 nm on a microplatereader (BioRad, iMark Microplate reader).

Wild type C2 showed clear binding, and loss of binding was only observedfor C2-(FB-S2) in which the complement C2 S2 domain (SEQ ID NO: 46) wasreplaced by the Factor B S2 domain (SEQ ID NO: 54). In contrast, nobinding was seen to wild type FB, however strong binding was detectedfor the mutant FB-(C2-S2) in which the Factor B S2 domain (SEQ ID NO:54) was replaced by the complement C2 S2 domain (SEQ ID NO: 46), asdepicted in FIG. 12. These results clearly show that anti-C2-5F2.4recognizes an epitope on S2 (Sushi domain 2) on C2b. This result alsoshows that C2-(FB-Pep1) is produced in sufficient quantity. Similarresults were obtained when using the mouse IgG2a anti-C2-5F2.4. Inaddition, similar results were obtained when binding was studied in thepresence of 1.25 mM Ca⁺⁺ in the buffer. Epitope mapping performed byBioceros BV, using domain swap mutants between human C2 and mouse C2,also led to a similar conclusion. Furthermore, the amino acid sequenceof Sushi domain 2 of cynomolgus C2 is completely identical to Sushidomain 2 of human C2.

Example 7: Fine Mapping of Epitope of Anti-C2-5F2.4 within Sushi Domain2

Anti-C2-5F2.4 does not cross-react with mouse C2, and the mouse S2domain (SEQ ID NO: 58) differs from the human S2 domain (SEQ ID NO: 46)at 10 amino acid positions, as depicted in FIG. 13. To investigate whichof these ten amino acids is responsible for the mAb binding, finemapping mutants were generated. The fine mapping constructs containedeither the human C2b fragment (huC2b), huC2b with mouse S2 (huC2b-mS2),and ten mutants, each containing one amino acid back-mutation from themouse sequence to the human sequence. Mutant C2b proteins were generatedsimilar to the domain swap mutants by transient transfection into HEK293cells.

All mutants were produced and detected by anti-FLAG ELISA, as depictedin FIG. 14. Anti-C2-5F2.4 bound to huC2b but not to huC2b with a mouseS2 (huC2b-mS2), as expected. None of the reverse point mutationsrestored binding of anti-C2-5F2.4, suggesting that the epitope of thismAb is composed of at least two amino acids on the S2 domain, asdepicted in FIG. 15. Similar results were obtained when binding wasstudied in the presence of 1.25 mM Ca⁺⁺ in the buffer.

By using the publicly available structural data for human C2b, theposition of the ten possible amino acids that might contribute to theepitope of anti-C2-5F2.4 was analyzed, as depicted in FIG. 16. Thisanalysis revealed three possible clusters, each composed of three aminoacids that could contribute to the epitope. DNA constructs for thesecluster mutants were designed and obtained. The cluster mutants weregenerated by mutating the human C2b S2 amino acids to correspondingmouse C2b S2 amino acids. In each mutant three amino acids were changed.A loss of binding was expected if these three amino acids contributed tothe epitope of anti-C2-5F2.4. FIG. 17A shows that cluster 1 mutant wasexpressed well and the binding was not affected, and therefore theseamino acids do not contribute to the binding. Based on the anti-FLAGELISA, expression of the cluster 2 mutant was lower, and this resultedin lack of binding by anti-C2-5F2.4. Cluster 3 mutant also was notexpressed well, and this most likely explains the lack of binding byanti-C2-5F2.4, as depicted in FIG. 17B. Similar results were obtainedwhen the binding was studied in the presence of 1.25 mM Ca⁺⁺ in thebuffer. From this analysis, the amino acids in cluster 1 can beexcluded, leaving four possible amino acids in cluster 2 and cluster 3.

Domain swap mutants provided strong evidence that the epitope that isrecognized by anti-C2-5F2.4 on C2 is located on the Sushi domain 2 onC2b. Additionally, these experiments suggest that the presence of thatdomain on FB is sufficient for recognition by anti-C2-5F2.4. Consideringthat anti-C2-5F2.4 does not react with mouse C2, one or more of the 10amino acids that differ between human and mouse Sushi 2 domain should beessential for the epitope. By using single amino acid back-mutations, weshow that a single amino acid in Sushi 2 cannot restore binding. Fromthe experiments performed with the cluster mutants it was concluded thatamino acids in cluster 1 do not contribute to the epitope ofanti-C2-5F2.4. Amino acids of cluster 2 may contribute to the epitope ofanti-C2-5F2.4, but since the expression of this mutant was lower thancluster 1 mutant, it cannot be excluded that the folding of cluster 2mutant was not optimal. Since the cluster 3 mutant was not wellexpressed, it appears likely the mutations affected folding and so therole of the amino acids in cluster 3 remains elusive.

INCORPORATION BY REFERENCE

All publications and patent documents cited herein are incorporatedherein by reference in their entirety.

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to human complement factor C2, wherein said monoclonal antibody or fragment thereof comprises: a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 3; and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:
 2. 2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a full-length monoclonal antibody.
 3. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody comprises a human IgG heavy chain constant domain.
 4. The monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the heavy chain constant domain comprises a human IgG1 heavy chain constant domain.
 5. The monoclonal antibody or antigen-binding fragment thereof of claim 4, wherein the human IgG1 heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:
 4. 6. The monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the heavy chain constant domain comprises a human IgG4 heavy chain constant domain.
 7. The monoclonal antibody or antigen-binding fragment thereof of claim 6, wherein the human IgG4 heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:
 5. 8. The monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the monoclonal antibody comprises a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 6 and a light chain comprising the amino acid sequence set forth as SEQ ID NO:
 7. 9. The monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the monoclonal antibody comprises a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 8 and a light chain comprising the amino acid sequence set forth as SEQ ID NO:
 7. 10. A pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human complement factor C2, and a pharmaceutically acceptable carrier, wherein said monoclonal antibody or fragment thereof comprises: a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 3; and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:
 2. 11. The pharmaceutical composition of claim 10, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a full-length monoclonal antibody.
 12. The pharmaceutical composition of claim 10, wherein the monoclonal antibody comprises a human IgG heavy chain constant domain.
 13. A nucleic acid molecule or plurality of nucleic acid molecules encoding the monoclonal antibody or antigen-binding fragment thereof of claim
 1. 14. A vector or plurality of vectors comprising the nucleic acid molecule or plurality of nucleic acid molecules of claim
 13. 15. A host cell comprising the nucleic acid molecule or plurality of nucleic acid molecules of claim
 13. 16. A host cell comprising the vector or plurality of vectors of claim
 14. 17. The host cell of claim 15, wherein the host cell is a mammalian cell.
 18. A method of making a monoclonal antibody or antigen-binding fragment thereof, comprising culturing a population of host cells of claim 15 under conditions suitable for expression of the monoclonal antibody or antigen-binding fragment thereof; and isolating the monoclonal antibody or antigen-binding fragment from the cells.
 19. A method of inhibiting classical pathway of complement activation in a subject, comprising administering to a subject in need thereof an effective amount of the monoclonal antibody or antigen-binding fragment thereof of claim
 1. 20. A method of inhibiting lectin pathway of complement activation in a subject, comprising administering to a subject in need thereof an effective amount of the monoclonal antibody or antigen-binding fragment thereof of claim
 1. 21. The method of claim 19, wherein the subject is a human. 